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Lecture Notes
Michael Palmer
Department of Chemistry
University of Waterloo
Waterloo, Ontario, Canada
The Logo
2
UNIVERSITY OF WATERLOO VISUAL IDENTITY GUIDE 2008 |
These notes are for use with my 3rd year undergraduate class on metabolism.
The focus is on human metabolism, and more specifically on human energy me-
tabolism. Apart from the pathways and reactions, several aspects of hormonal
regulation and some medical correlations are also included. The entire text
has been prepared by myself. The same applies to most figures; exceptions are
indicated. With respect to my own material, you are welcome to use it in your
own not-for-profit teaching materials as you see fit, provided that proper credit
is given.
These notes are currently in their 4th edition. In this edition, some errors
have been corrected, yet likely some more remain to be discovered, and new
ones may have been introduced. I therefore welcome corrections and sug-
gestions for improvement, no matter whether you’re a colleague, a student,
or simply an interested reader. Please send email to mpalmer@uwaterloo.ca.
Thank you.
Contents
Chapter 1 Introduction 1
1.1 Motivation: Why would you study metabolism? . . . . . . . . . . 1
1.2 Catabolic and anabolic reactions . . . . . . . . . . . . . . . . . . . 1
1.3 Metabolic diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.4 Types of foodstuffs . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.5 The digestive tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.6 What’s next? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Chapter 2 Refresher 16
2.1 How enzymes work: Active sites and catalytic mechanisms . . 16
2.2 Classification of Enzymes and enzyme reactions . . . . . . . . . 18
2.3 Energetics of enzyme-catalyzed reactions . . . . . . . . . . . . . 19
2.4 The role of ATP in enzyme-catalyzed reactions . . . . . . . . . . 21
2.5 Regulation of enzyme activity . . . . . . . . . . . . . . . . . . . . 23
Chapter 3 Glycolysis 26
3.1 Overview of glucose metabolism . . . . . . . . . . . . . . . . . . . 26
3.2 The place of glycolysis in glucose degradation . . . . . . . . . . 27
3.3 Reactions in glycolysis . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.4 Mechanisms of enzyme catalysis in glycolysis . . . . . . . . . . 28
3.5 Energy-rich functional groups in substrates of glycolysis . . . . 35
3.6 Function of glycolysis under anaerobic conditions . . . . . . . . 36
3.7 Transport and utilization of glucose . . . . . . . . . . . . . . . . 37
iii
iv CONTENTS
Chapter 7 Gluconeogenesis 85
7.1 Reactions in gluconeogenesis . . . . . . . . . . . . . . . . . . . . . 86
7.2 Glucogenic amino acids and gluconeogenesis . . . . . . . . . . . 88
7.3 Regulation of gluconeogenesis . . . . . . . . . . . . . . . . . . . . 88
7.4 Energy balance of gluconeogenesis . . . . . . . . . . . . . . . . . 92
7.5 The Cori cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
7.6 The glyoxylate cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
7.7 Additional roles of gluconeogenetic enzymes . . . . . . . . . . . 94
Introduction
1
2 1 Introduction
complex
biomolecules ADP+Pi
NADP+ O2
NADPH+H+
ATP
small intermediates CO2 + H2O
Figure 1.1 A broad outline of human metabolism. Foodstuffs, which are mostly ma-
cromolecules, are broken down in catabolic reactions to small intermediates, which in
turn are in part further degraded to yield energy (ATP) and reducing power (NADPH).
Some of the intermediates thus produced are used in anabolic reactions for the synthe-
sis of new macromolecules. These may in turn be recycled and used as substrates for
the release of small intermediates.
Ribose 5-P
Fructose 1-P Glyceraldehyde Glyceraldehyde 3-P/
Dihydroxyacetone-P
1,3-bis-P-glycerate
Glycerol-P Glycerol
3-P-glycerate
Triacylglycerol
2-P-glycerate
Figure 1.2 Metabolic pathways covered in this class. Solid arrows indicate single
enzyme reactions, with the exception of the respiratory chain, which converts H2 to
H2 O. Dashed arrows represent sequences of enzyme reactions.
4 1 Introduction
The sum formulas of sugars can approximately be written as (CH2 O)n , that
is they formally are multiples of carbon (C) plus water (H2 O). They are therefore
collectively referred to as carbohydrates. These form an important part of our
diet. Indeed, in the typical diets of many countries, the most calories by far are
derived from carbohydrates, contained for example in rice, wheat, or potatoes.
To the right of the center in Figure 1.2 you will find triacylglycerol. This is
another major component in our diet: Fat. The decomposition of fat yields fatty
acids as the main component; these are further degraded in the β-oxidation
pathway to acetyl-CoA. Acetyl-CoA is also an intermediate in the complete
degradation of carbohydrates and of proteins. Therefore, acetyl-CoA is a cen-
tral hub in metabolism, through which all substrates destined for complete
oxidative degradation must pass. Acetyl-CoA is also a precursor in several
biosynthetic pathways (Figure 1.3b).
The third major type of foodstuff are proteins. Of the twenty standard
amino acids found in proteins, we can synthesize only ten ourselves; the other
ones are called essential amino acids. Therefore, while a low amount of dietary
carbohydrates or fat can be compensated by our own biosynthesis,1 lack of
dietary protein cannot. Accorgingly, in poor countries, lack of dietary protein
is the most common form of malnutrition.
One class of macromolecules that is not covered here are the nucleic acids.
Nevertheless, nucleic acids make up a sizeable fraction of our food. Only the
sugar part of each nucleotide—ribose or deoxyribose—can be utilized to gain
energy. The bases—A,C,G,T—can be used as building blocks for the synthe-
sis of new nucleotides and nucleic acids. If they are present in excess over
biosynthetic demands, they are modified and excreted.
Where does metabolism occur? The first step, the depolymerization of food-
stuff macromolecules, occurs extracellularly, certainly in humans but typically
even with organisms as simple as bacteria. Depolymerization is accomplished
by digestive enzymes, which are secreted by the cell, and uptake of substrates
into the cell only occurs at the stage of the monomeric breakdown products.
Why is that so? Extracellular digestion makes the substrates available to ev-
eryone, not just to the cell that has provided the necessary enzymes. It is
therefore a potentially ineffective process, especially with unicellular organ-
isms that often will find themselves competing for substrates with numerous
other species.
1 The traditional diet of eskimos, for example, is extremely low in carbohydrates, since it
a) Foodstuffs
Fatty acids,
Fat CO2 + H2O
glycerol
Urea, sulfate
ADP ATP
Acetyl-CoA CO + H O
2 2
An obvious answer is that there are no transport mechanisms for the uptake
of macromolules across the cell wall. While that is true,2 there is a deeper
reason – taking up macromolecules in a non-specific way would open the door
for all kinds of Trojan horses. Extracellular digestion is a kind of a firewall
to exclude hazardous biomolecules.3 Extracellular depolymerization is also
the strategy employed by our own digestive tract (Figure 1.4). The digestive
2 But not universally – for example, bacterial cells have evolved mechanisms for the active
uptake of DNA, which they switch on under special circumstances. This is exploited in the lab in
transformation experiments.
3 Amoebae, for example, do indeed ingest not only macromolecules but even whole bacteria.
They can do so because they confine the ingested material within phagosomes, which they swiftly
flood with acid and aggressive chemicals and enzymes to kill and degrade the bacteria. The same
occurs in our phagocytes, which are an essential part of our immune system.
1.5 The digestive tract 7
a) b)
stomach
liver
liver
pancreas
bile
stomach
bladder
pancreas
pancreatic duct
duodenum
Figure 1.4 Schematic of the digestive tract. a: Overview. b: The liver and the pancreas
both have secretory ducts that discharge into the duodenum, which is the topmost part
of the small intestine.
tract contains specialized organs and cells for the secretion of depolymerizing
enzymes, for the performance of the digestion, and finally the uptake of the
released substrates.
Pepsin
pH 2
Trypsin etc.
pH 7.5
Figure 1.5 Digestion of proteins. The acidic pH in the stomach causes the proteins
to unfold, which makes the peptide bonds accessible to pepsin. The fragments will not
be able to refold, even though the pH reverts to neutral in the small intestine, so that
the digestion can be completed, and the final products be taken up.
that these viruses are able to traverse the stomach and then infect the intestinal
mucosa.
O
HC
H OH
CH 2OH CH 2OH
OH
O HO H O
OH OH
H OH
OH OH OH
OH H OH OH
H OH
H
… HO
OH
O
O
OH
O
O
OH
O
O
OH
O
O
OH OH OH
H CH 2OH
OH branch
CH 2OH CH 2OH CH 2OH CH 2
O O O O O O
OH OH OH OH OH
O O O O O O
OH OH OH OH OH OH
Amylopectin
CH 2OH CH 2OH
O O
OH OH
OH O OH
OH OH
Maltose
Figure 1.6 Structures of glucose, amylose and maltose. Top: Glucose occurs in two
anomeric ring forms, as well in an open chain form. Middle: Amylopectin is a branched
polymer of α-glucose. The unbranched polymer is called amylose; both are contained
in starch. Bottom: Maltose is the disaccharide which results from the degradation of
amylose by amylase. It is in turn cleaved to glucose by maltase at the surface of the
intestinal mucosa.
Among its many other functions, the liver also serves as an exocrine gland.
The digestive juice secreted by the liver is known as the bile and is rich in bile
acids (see Figure 11.1), which are important in solubilizing fat so as to render it
10 1 Introduction
After digestion, the metabolites have to be taken up by the cells at the sur-
face of the mucosa of the small intestine, which comprises the duodenum,
the jejunum, and the ileumsmall intestinesections of. How does this uptake
work? Most nutrients are taken up by active transport, which can transport
solutes energetically uphill, that is against their concentration gradients. Active
transport is necessary for the quantitative uptake of the nutrients. An impor-
tant example is the transport of glucose, the most important end product of
carbohydrate digestion. The uptake of glucose is driven by the simultaneous
uptake of two sodium ions per molecule of glucose; this coupling is effected
by the SGLT1 transporter (Figure 1.7). While sodium is plentiful in the gut
lumen, its concentration is low inside the cells. The uphill transport of glucose
is therefore driven by the simultaneous downhill movement of sodium. Similar
transporters exist for other sugars (e.g. galactose) and for amino acids.
The amount of nutrient substrates that have to pass across the surface of
the gut is considerable; therefore, a large active surface is required. You have
probably heard that the length of the small intestine is likewise considerable –
about twice the height of your body. In addition, the inner surface of the small
intestine is folded, and the folds in turn have a shaggy surface, as have the
individual cells. All these factors combine to maximize the effective surface
available for substrate uptake. This is illustrated in Figure 1.8.
All digestion and nutrient absorption occurs in the small intestine, mostly in its
upper portions. In fact, even the lower segments of the small intestine usually
don’t get to do much work in nutrient uptake but have a more specialized role
in the reuptake of bile acids, and of things such as vitamin B12 , the lack of
which causes pernicious anemia. In the large intestine, essentially no nutrients
are left for absorption. What, then, is the function of the large intestine? A
4 Bile acids solubilize fat because they are detergents, and as such they are also useful for
2 Na+ 2 Na+
SGLT1 GLUT2
Figure 1.7 Mechanism of glucose uptake from the gut lumen. The transport against
the concentration gradient is powered by cotransport of sodium ions via the trans-
porter SGLT1 (SGLT=sodium glucose transporter), while the transport into the blood is
down a concentration gradient and can therefore be accomplished by simple facilitated
diffusion through GLUT2 (GLUT=glucose transporter).
key aspect of the large intestine is that it is inhabited by the so-called gut flora,
which comprises literally trillions of bacteria.5 What are these guys doing there,
other than producing foul smells, and why do we afford them such generous
shelter? There are several benefits of this:
1. Some bacteria are capable of synthesizing some vitamins, such as folic
acid, the lack of which again causes anemia, with symptoms and laboratory
findings very similar to those observed when vitamin B12 is lacking. Both folic
acid and vitamin B12 are required in the synthesis of nucleic acids. Inhibition
of DNA synthesis has the strongest impact in rapidly proliferating tissues such
as the bone marrow, which is the site of blood cell regeneration.
2. Our diet contains some substrates such as strange sugars and polysac-
charides that we cannot utilize or absorb, and which therefore travel through
the small intestine unaltered. Some bacteria can break down these substrates.6
Since the milieu in the colon is anaerobic—that is, it lacks oxygen—breakdown
will not be complete. Fairly common end products of fermentation are small
acids such as acetic or propionic acid7 Some of these products are then taken
up across the colon epithelium and utilized in our metabolism.
5 In fact, the number of bacteria in our large intestine is higher than that of our own body cells!
well as amino acids is in fact a traditional methodology for classifying enteric bacteria.
7 Our good old friend Escherichia coli and other bacteria also produce formic acid, which they
villi
microvilli
Figure 1.8 Structure of the mucosa of the small intestine. Top: Schematic, show-
ing the surface structure at increasing power of magnification. The inner sur-
face of the intestine is folded, and the folds in turn have a shaggy appearance
because the mucosa is folded up again into villi. The epithelial cells that consti-
tute the mucosal surface are in turn covered by microvilli. Bottom: Villi, shown
in a stained section of the intestinal mucosa (light microscopy, left), and microvilli
on the surface of an epithelial cell (scanning electron microscopy). Below the sur-
face, you can see the folded membranes of the endoplasmic reticulum. The mi-
croscopic images have been reproduced, with permission from Roger Wagner, from
http://www.udel.edu/Biology/Wags/histopage/histopage.htm.
Systemic
circulation
Liver vein
Liver
Figure 1.9 The portal circulation. The intestines receive arterial blood from the heart,
just like any other organ. The venous blood, however, is not passed back directly to
the right heart, as is the case with practically all other organs, but first reaches the liver
through the portal vein. The liver also receives a direct supply of oxygen-rich blood
through the liver artery. After passage through the liver, the blood received through
both the portal vein and the liver artery is fed into the general circulation.
the end of the small intestine, 5-5.5 liters have been reclaimed. Recovery of
this residual amount is hampered by the fact that the non-utilized solutes are
osmotically active, that is they will hold on to water and drag it down the pipe.
Utilization of these solutes in bacterial metabolism reduces the osmotic activity
of the colon content, enabling a more complete reuptake of water.8
So, you see that our intestinal bacteria are actually quite useful. On the
other hand, the bacterial fermentation also produces ammonia and other toxic
products that must be captured and disposed of by the liver. This is not a
problem in healthy individuals but can become one in persons suffering from
liver disease.
Upon uptake, most solutes will be exported on the other side of the mucosal
cells and then find themselves in the blood stream. A peculiarity of the in-
testines consists in the fact that all blood drained from them is first passed
8 On the other hand, increasing the osmotic activity of the gut content is a straightforward
way of limiting water reuptake in order to treat constipation. A traditional drug based on this
principle is sodium sulfate (now out of fashion).
14 1 Introduction
on to the liver via the portal vein (Figure 1.9) portal circulation before being
released into the general circulation. This serves a twofold purpose:
1. It gives the liver a chance to take excess amounts of substrates—glucose,
amino acids—out of circulation and to store and process them. This serves
to maintain stable blood nutrient concentrations, which is important for the
well-being of the more sensitive and fastidious cells in the rest of the body.
2. The bacteria in the large intestine produce ammonia and other toxic
metabolites, which are cleared by the liver. In patients with liver failure, these
toxic metabolites spill over into the systemic circulation, which will among
other things lead to disturbances of cerebral function. This activity of the liver
also affects many drugs; the inactivation of drugs by the liver immediately
following intestinal uptake is known as the first pass effect.9
The liver has a particular tissue structure that enables it to exchange solutes
with the blood very efficiently. While in most organs the blood is contained in
blood vessels with clearly defined boundaries and walls, the liver has a sponge-
like structure that permits direct contact of the blood plasma with the liver
cells (Figure 1.10).
To liver
vein
b)
Figure 1.10 Blood circulation and tissue perfusion in the liver. a: The liver tissue
has a honeycomb structure; each hexagon constitutes one liver lobule. The liver artery
and portal vein branches are located at the corners of each lobule. The blood seeps
out of the artery and portal vein branches, into the sinusoids of the liver lobule. It
is collected by the lobule’s central vein, which drains it toward the liver vein and the
general circulation. b: Higher power view, showing the sponge-like structure of the
liver tissue. The liver cells are stained in brown; the white space in between are the
sinusoids. While in the sinusoids, the blood gains surrounds virtually every liver cell.
The microscopic images have been reproduced, with permission from Roger Wagner,
from http://www.udel.edu/Biology/Wags/histopage/histopage.htm.
and it bypasses the liver because it is delivered into the lymphatic vessels rather
than into the blood stream.
We will consider all these processes in turn. At the end of this class, we will
not only have gained a solid grasp of these pathways, but we will also have the
tools to understand in depth what goes wrong in metabolic diseases such as
phenylketonuria, lactose intolerance, and diabetes mellitus.
Chapter 2
Refresher
This chapter attempts to summarize some essential concepts from second year
biochemistry. Feel free to skip it if you remember a thing or two from that
distant past.
16
2.1 How enzymes work: Active sites and catalytic mechanisms 17
a) O R2 O R4
H
H 2N N
N N
H H
R1 O R3 O
b) O
O
Ser 195
H 2N
H2N
O Attack on Asp 102
O
H substrate
H peptide bond
N
N His 57
His 57
N
N H H 2N
H2N
H O O Ser 195
O O
H2N
H 2N O
O
Asp 102
O
O
c) A
A−
R2 O
H
H H O
R N R2 O
R2
N C R H
H R N N
R O H R
R3 N C R
O H N C
O O H R3
O R3
O
Ser−Enzyme Ser−Enzyme Ser−Enzyme
O H
d) O OH
R2 H
H R2 O
R R2 H O
N H
R R N
N C R
H N C R N
H N C O H R
O R3 HO H
O R3
OH OH R3
Figure 2.1 Structure and mechanism of chymotrypsin. a: The peptide bond cleaved
by chymotrypsin. b: The interplay of the side chains of histidine 57, aspartate 102, and
serine 195 that results in the deprotonation of serine. All three side chains are close to
each other in the active site of the enzyme. c: The enzyme- catalized reaction. d: The
base-catalyzed reaction for comparison.
tract, where its job is to knock down large protein molecules into small peptides
that are then further processed by peptidases.
1 You will note that, nevertheless, many textbook figures give the enzyme structures as ribbon
diagrams that just show the fold of the protein backbone but omit the side chains. Therefore,
these diagrams are completely useless for understanding the catalytic mechanism of an enzyme.
18 2 Refresher
Barrage
Turbine
the reactants. The very short-lived, energy-rich state at the top of this barrier is
called the transition state. Enzymes can make a big difference to the activation
energy ∆G∗ and thus accelerate reactions, but they cannot change the free
energy ∆G—and therefore, the direction—of the reaction.
2.3.1 A simile
4 Der See=German for the lake, die See=German for the sea
2.4 The role of ATP in enzyme-catalyzed reactions 21
• Like the conduits, enzymes can facilitate the flow and (typically) control
its rate but not its direction. The direction of the flow will depend solely
on the difference in altitude (∆G).
It is usually best not to take analogies too far, because otherwise they be-
come obfuscating rather than enlightening. Here are some dissimilarities:
• All tunnels are basically alike. In contrast, each enzyme needs a specific
“trick” or catalytic mechanism suited to the specific metabolites it is deal-
ing with. Investigating the catalytic mechanisms of individual enzymes is
an important part of biochemistry.
• The energy level of a pool in the hydro-electric system is completely
described by its altitude. However, the free energy of a metabolite is
dependent on its concentration; the lower the concentration, the lower
the free energy.
• Although enzymes cannot revert an endergonic reaction (that is, a reaction
with ∆G > 0) in isolation, they can make it go uphill by coupling it to
another one that is exergonic, so that the overall ∆G becomes negative.
• Finally, water will never spontaneously flow uphill – molecules, however,
do occasionally move spontaneously to somewhat higher energy levels.
If molecules are offered two states with different energy levels, such as two
different conformations or two different tautomeric states, they will sponta-
neously distribute between them and establish equilibrium. In equilibrium,
the relative occupancy of these two states will solely depend on the energy
difference between them:
n1
= e− RT
∆G
(2.1)
n2
where n1 and n2 represent the numbers of molecules in the high and low
energy states, respectively. (R is the gas constant, whereas T is the absolute
temperature.)
The above equation applies to the distribution of the initial and the final
states of a reaction. It also applies to the distribution between molecules be-
tween the initial state and the transition state of a reaction. The tendency of
molecules to spontaneously assume states of higher energy explains that chem-
ical reactions can occur at all, even though the energy level of the transition
state is always higher than those of the initial and final states. However, the
higher the activation energy, the more rarified the transition state will become.
The number of molecules that can make it across the barrier thus becomes
smaller, and the reaction slower with increasing activation energy.
O O
H2N CH
CH2 + O O O
CH2 O P O P O P O Adenosine
O O O
O O
HO O
H2N CH O O
CH2 + O P O P O Adenosine
CH2 O O
O
O O P O
O
NH3
HO O O O
+ O P O P O Adenosine
H2N CH
O O
CH2
CH2 O
+ O P O
O NH2
O
Figure 2.3 The catalytic mechanism of glutamine synthetase. The terminal phos-
phate is first transferred to glutamate to form a carboxyphosphate. In this energy-rich
intermediate, the phosphate is a good leaving group and is easily substituted by am-
monia.
ATP (cosubstrate)
Fructose-6-P
ADP (regulator)
a) c)
R A R A
Active Inactive
conformation conformation
d)
P
Allosteric Allosteric
activation inhibition
Inhibition by phosphorylation
Glycolysis
Glucose is a key metabolite in human metabolism, and we will spend a good bit
of time on the several pathways that are concerned with the utilization, storage,
and regeneration of glucose.
muscle tissue is smooth muscle, which occurs in blood vessels and internal organs and is under
autonomous control.
26
3.2 The place of glycolysis in glucose degradation 27
1 2 3 Fatty acids,
Glucose Pyruvate Acetyl-CoA
triacylglycerol
H2O
4
CO2
“H2“
O2 ADP + Pi
5
ATP
H2O
Figure 3.1 gives an overview of the steps involved in the complete degradation
of glucose. It is evident that, in the overall process, glycolysis is only the
first step. The pyruvate it generates is turned into acetyl-CoA by pyruvate
dehydrogenase. Acetyl-CoA is completely degraded in the citric acid cycle and
the respiratory chain. While some ATP is generated at each of these stages,
most of it is produced in the respiratory chain.
If glucose is available in excess of immediate needs and of glycogen storage
capacity, it will still be broken down by glycolysis and pyruvate dehydrogenase
to acetyl-CoA. However, acetyl-CoA will then be fed into fatty acid synthesis,
which occurs in the liver and the fat tissue.
28 3 Glycolysis
3.4.1 Hexokinase
Our first example is hexokinase, which carries out the first reaction in the gly-
colytic pathway. This type of reaction—the transfer of the terminal phosphate
group from ATP onto a hydroxyl group on the substrate—is a very common
reaction in biochemistry, and we will see many more examples in this class.
The mechanism of phosphorylation is always the same, so it suffices to discuss
it once.
Most reactions that involve the transfer of a phosphate group from ATP
to something else are exergonic, that is they are energetically favourable. For
example, the hydrolysis of ATP—which is the transfer of the phosphate group
3.4 Mechanisms of enzyme catalysis in glycolysis 29
O
Glucose Glucose-6-P Fructose-6-P
HO P O-
O
HO O HO P O-
O OH
O ATP ADP O O
OH OH
OH
OH OH OH OH OH
OH 1 OH 2 OH
ATP
1,3-Bisphosphoglycerate
O
3
ADP
HO P O- GAD-3-P DHAP
O O OH
O O O
+ +
OH NADH+H NAD OH O HO P O- HO P O-
+ O O
O
O 6 O O 4
Pi OH
HO P O- HO P O- HO P O- OH
O O O OH
5
Fructose-1,6-bis-P
ADP
7
ATP
2-Phosphoglycerate Phosphoenolpyruvate Pyruvate
O -
O -
O - O-
O O O -
O O -
O
10
OH O P O O P O O
8 OH 9 OH
ADP ATP
O OH
HO P O-
O 3-Phosphoglycerate
a) O O O
b)
O P O P O P O Adenosine
Arg
R X O O O Lys HN NH
Mg++ +
NH3
+
O O O NH3 O O O
R X P O O P O P O Adenosine O P O P O P O Adenosine
O O O R X O O O
Figure 3.3 Mechanism of phosphate group transfer from ATP. a: The phosphorus in
the center of the terminal phosphate group is attacked by a nucleophile, which often is
an anion (R–X– ) but can also be a free electron pair. Attack is inhibited by the negative
charges on the oxygen atoms around the phosphorus. This electrostatic repulsion
is responsible for the high activation energy barrier of the uncatalyzed reaction. b:
ATP is activated towards nucleophilic attack through electrostatic shielding both by
magnesium and by positively charged amino acid side chains in the active site of the
enzyme.
to water—has a free energy of −35 J/K mol. It is interesting to note then that,
despite the abundance of water, ATP is quite stable in solution. This indicates
that there must be a high activation energy barrier that resists cleavage of the
phosphodiester bond.2
This energy barrier is due to the negative charges within the phosphate
group that shield the central phosphorus from nucleophiles that likewise are
negatively charged. Accordingly, to lower this barrier, kinases provide com-
pensating positive charges within the active sites, which engage the negative
charges on the ATP molecule and thereby facilitate nucleophilic attack (Figure
3.3).
The intracellular concentration of glucose will be in the low millimolar range,
whereas water is present at a concentration of more then 30 mol/l. Apart from
the task of activating ATP, then, hexokinase also must ensure that the activated
ATP does indeed react with the hydroxyl group on the glucose molecule but
not of water. How is this specificity accomplished?
Like many other enzymes, hexokinase exists in an open and a closed confor-
mation. It binds its substrate and cosubstrate, glucose and ATP, in the open
state, whereupon it changes its conformation to the closed state. This change
is necessary for the enzyme’s catalytic activity, and it also is accompanied by
the expulsion of water from the active site (Figure 3.4). In this way, the only
hydroxyl group that remains in the vicinity of the activated ATP will be that
on the C6 of glucose. However, if we offer the enzyme xylose as a substrate
2 If you are unclear about the difference between free energy and activation energy of a chemical
a) b) H2C OH
O
OH
OH OH
Glucose OH
H
OH
O
OH
OH OH
OH
Xylose + water
Figure 3.4 How hexokinase circumvents ATP hydrolysis. a: After binding of its
substrate glucose (red/spacefill) and its cosubstrate ATP (not shown), hexokinase
(blue/wire- frame) adopts a closed conformation, in which substrate and cosubstrate
are buried within the enzyme. In this way, water is excluded from the active site and,
hence, from the reaction. b: If xylose is used instead of glucose, one water molecule can
squeeze along with it into the active site and then react with ATP, leading to hydrolysis.
instead of glucose, we can fool it into hydrolyzing ATP rather than phospho-
rylating the sugar. Xylose looks exactly like glucose with respect to the ring
(C1 – C5), so it is able to bind to the active site of hexokinase and induce the
activating conformational change. However, because it lacks the C6 atom with
its attached hydroxyl group, it leaves space within the active site for one water
molecule (Figure 3.4b), and it is this water molecule that will react with the
activated ATP in this case.
CH2O-Pho. Glucose-6-P
HC O
2
HC OH CH B+
H CH2O-Pho.
OH C C OH H
H HC O
OH
HC OH CH
1 H 3
B+ OH C
H
C O H B:
CH2O-Pho
OH
H - HC OH
B H H
HC OH C
OH C O
H
OH H+
- -
B B
5 4
:B CH2O-Pho
B:
CH2O-Pho
HC O H HC OH
H2 H
HC OH HC OH C
C
CH2O-Pho O
OH C OH OH C
H H+
H
HC O
H2 O O H
H
HC OH C H B B
OH C OH
H
OH Fructose-6-P
Figure 3.5 The catalytic mechanism of phosphohexose isomerase. 1: The active site
contains 2 bases in strategic positions. 2: Glucose-6-phosphate bound the active site,
and the ‘electron-hopping’ steps that lead to ring opening. 3-5: Subsequent reaction
steps that lead to migration of the keto group and ring closure of fructose-6-phosphate.
ple in the citric acid cycle and in the β-oxidation of fatty acids. The reaction
goes through the following steps (Figure 3.6):
1. The substrate binds to the active site, where NAD+ is already bound.
2. A deprotonated cysteine thiol group (–S– ) in the active site performs a
nucleophilic attack on the aldehyde carbon, which yields a tetrahedral
intermediate state in which the enzyme and the substrate are covalently
bound to each other – hence the term “covalent catalysis”.
3. The intermediate transfers 2 electrons and a proton to NAD+ , yielding
NADH and a thioester.
4. NADH leaves and is replaced by NAD+ .
5. The thioester is cleaved by a phosphate ion, again by nucleophilic attack.
6. The product (1,3-bisphosphoglycerate) leaves, and the enzyme is restored
to its original state.
The redox cosubstrate used by glyceraldehyde-3-phosphate dehydrogenase,
nicotinamide adenine dinucleotide (NAD+ ), is the major acceptor of hydrogen
3.4 Mechanisms of enzyme catalysis in glycolysis 33
a) O O O O
P P
H O O O
H O O O O O
OH R OH R
O O
O P O O P O
O O
b)
1 2 3
NAD+ NAD+ H NAD
+
H O
H O
- - C O
S S S
R R
R
+ + +
HB HB HB
6 5 4
+ + NADH
NAD
NAD
O O O
C C +
S—H S O P OH S NAD
R R
O
:B + +
HB HB
O
O
O P OH
R O
abstracted from substrates throughout glycolysis and the citric acid cycle. Its
structure and its reduction by hydrogen are shown in Figure 3.7. As you can
see, all the action occurs at the nicotinamide moiety – the adenosine part is
completely out of the picture, as far as the redox chemistry is concerned. Why,
then, is it there at all? One answer is that it serves as a “tag”, an identifier
that enables the coenzyme to interact with a defined set of enzymes. There
34 3 Glycolysis
a) O b)
NH2 O
+ E S C H
N O
R H
C O C
HC NH2
O
HC +
CH
O P O N
OH OH
O R
NH2
O P O
O
O N
N E S C
H H O
C N R
O N
HC NH2
HC CH
N
OH OR R=H: NAD+
R=phosphate: NADP+ R
is a second, very similar coenzyme, NADP+ , which performs the same redox
chemistry but has a different tag with an extra phosphate group and is used by
a different set of enzymes. The reasons for this are discussed in a later chapter
(see section 9.3).
It is also interesting to note that, as in many other cosubstrates, the tag
consists of a nucleotide instead of a peptide. This likely hearkens back to
the ancient RNA world, in which all catalytic functions are supposed to have
been performed by RNA enzymes instead of proteins. These RNA enzymes are,
for the most part, now extinct, having been replaced by presumably superior
protein enzymes. However, cosubstrates can’t evolve as easily as enzymes,
since they interact with many different enzymes and are therefore are subject
to many more evolutionary constraints; they thus remain frozen in time, like
molecular fossils.
NH2
N
N
O O O
O P O O P O P O C N
O N
HO O + O O
H
CH2
O
OH OH
HO O H HO O
CH2 CH3
O O
Figure 3.8 Mechanism of the pyruvate kinase reaction. The enolpyruvate formed
intermittently tautomerizes to pyruvate; it is this step that drives the reaction in the
indicated direction.
in turn is driven by the partial oxidation of carbon 1, which changes from the
aldehyde form to the more highly oxidized carboxylate form.
In summary, the following functional groups can provide sufficient energy
to drive the synthesis of ATP:
1. thioesters – when cleaved,
2. aldehydes – when oxidized to carboxylic acids,
3. carboxyphosphates – when cleaved, and
4. enolphosphates – when cleaved.
the capacity of a trained athlete to sustain aerobic rather than anaerobic metabolism during
prolonged exertion. The anatomical correlate of endurance is not so much the build-up of muscle
tissue but the extent of its vascularization, that is the abundance of capillaries in the tissue. A
high density of capillaries ensures good oxygen supply.
3.7 Transport and utilization of glucose 37
a) Pyruvate Lactate
O OH O OH
NAD+
NADH+H+
O HC OH
CH3 CH3
b) Pyruvate
O OH +
CO2 NADH+H+ NAD
O HC O H2C OH
Figure 3.9 Regeneration of NAD+ for glycolysis under anaerobic conditions. a: The
lactate dehydrogenase reaction is utilized by mammalians. b: Ethanolic fermentation
occurs in yeast. Pyruvate generated by glycolysis is decarboxylated to acetaldehyde.
Reduction of the latter regenerates NAD+ and yields ethanol, which is less toxic than
both the aldehyde and the lactic acid.
carry away, dilute and buffer the excess acid. One effective strategy, then, is to
chemically degrade it. This is the biological purpose of ethanolic fermentation
(Figure 3.9b).
+
Figure 3.10 Facilitated diffusion, a form of protein-mediated membrane transport.
Typically, substrate binding and dissociation are fast as compared to the conforma-
tional change in the transporter protein that leads to release of the substrate on the
far side of the membrane.
the kidney tubules, these transporter proteins operate not by active transport
but by facilitated diffusion, that is they simply speed up transport of glucose
along its concentration gradient (Figure 3.10). Although such transporters are
not enzymes, there is one similarity: With both enzyme reaction and with fa-
cilitated diffusion, substrate binding and dissociation are much faster than the
action of the protein – the enzyme reaction, or in this case the conformational
change required for translocation. Therefore, like simple enzymes, facilitated
transport obeys Michaelis-Menten kinetics:
[S]
V = Vmax (3.1)
KM + [S]
You may recall (or else infer from the above equation) that the affinity of the
protein for the substrate, which is represented by KM , controls the dependency
of the activity of on the substrate concentration. In most tissues, the glucose
transporters have a low KM , which means that each transporter molecule is
always operating at full speed,4 irrespective of the glucose concentration. In
contrast, the transporter subtype found in the liver (GLUT2) has a higher KM ,
so that the rate of transport is reduced at low glucose concentration.
A similar difference is observed at the stage of glucose phosphorylation.
The liver has a special enzyme called glucokinase, which performs the same
reaction as does hexokinase but differs from the latter by a higher KM value.
Accordingly, phosphorylation will proceed at reduced rate at a low level of
blood glucose (Figure 3.11), and most of the glucose will be allowed to pass
the liver and make its way into the general circulation. Conversely, at high
4 This does not mean that the overall extraction of glucose always goes at full speed in all
tissues. Regulation of glucose uptake in many tissues is mediated by insulin, which changes
the overall number of available transporters but not the activity of the individual transporter
molecule (See section 13.1.2).
3.7 Transport and utilization of glucose 39
Glucokinase
(liver)
V/Vmax
5 10 15 20 25
Glucose (mM)
Starch is the most important carbohydrate in our diet. As we have seen, starch
consists of glucose, which is therefore the most important dietary monosaccha-
ride. Other quantitatively important sugars are:
1. Lactose (milk sugar) is a disaccharide of glucose and galactose and is
found in milk (surprise!).
2. Sucrose is a disaccharide of glucose and fructose and is found in many
plants and fruit, most prominently in sugar cane and sugar beet.
3. Fructose, as the free monosaccharide, is also found in significant amounts
in the diet, both in fruit and as a sweetener.
4. Sorbitol is a sugar alcohol, that is the carbonyl group found in fructose or
glucose is reduced to a –CHOH group. It is used as a sweetener but also
occurs naturally in the diet.
5. Nucleic acids contain ribose and deoxyribose.
Ribose is part of the hexose monophosphate shunt and will be covered in the
corresponding chapter. Here, we will focus on the first three sugars.
The main motif in the metabolism of these sugars is economy: Instead of
completely separate degradative pathways, there are short adapter pathways
that funnel them into the main pathway of carbohydrate degradation, that is
glycolysis. An overview of these pathways is given in Figure 4.1.
40
4.1 Metabolism of sucrose and fructose 41
UDP-Galactose UDP-Glucose
Pyruvate
Sucrose is produced from sugar cane and sugar beet, where it is found in very
high concentrations (15-18%). In our “healthy” western diet, it may amount to
as much as 20% of dietary carbohydrate. Sucrose contains glucose and fructose
joined in a β-glycosidic bond (Figure 4.2).
Hydrolytic cleavage of sucrose, like that of of maltose, occurs at the surface
of the intestinal epithelial cells. The enzyme responsible is β-fructosidase, also
named sucrase. Both sugars are then taken up by specific transport: Glucose by
the SGLT1 transporter, and fructose by the GLUT5 transporter, which is named
after glucose but in fact is more active on fructose than on glucose.
Fructose degradation, sometimes called fructolysis, is carried out in the
liver. In the first step, fructose is phosphorylated by fructokinase, which uses
ATP as a cosubstrate. This yields fructose-1-phosphate. The latter is then
cleaved by aldolase B, which is found mainly in the liver, in keeping with the
liver’s prominent role in fructose degradation. The products of this reaction
are dihydroxyacetone phosphate, which is a metabolite in glycolysis, and glycer-
aldehyde. Finally, glyceraldehyde is phosphorylated by glyceraldehyde kinase.
This yields glyceraldehyde-3-phosphate, which again is an intermediate of gly-
colysis (Figure 4.3).
Glyceraldehyde can alternatively be utilized by conversion to glycerol and
then to glycerol-1-phosphate. The latter is a substrate in the synthesis of
triacylglycerol, that is fat. Fructose and sucrose appear to promote obesity
42 4 Catabolism of sugars other than glucose
CH2OH
CH2OH CH2OH
O OH O O
OH 2 OH 2 OH
CH2OH CH2OH
OH O
OH OH
OH
Figure 4.2 Structures of fructose (left) and sucrose. In the sucrose molecule, the
fructose is “standing on its head”, and it is the carbon No. 2 that is joined to the
carbon No. 1 of glucose. Glucose is in its α configuration, whereas fructose is in its β
configuration.
more strongly than equivalent amounts of starch or glucose; if that is the case,
the utilization via glycerol-1-phosphate may be among the reasons.
blood.
4.2 Metabolism of lactose and galactose 43
O
Dihydroxyacetone−P
CH2OH H2C OH
O CH2OH ATP ADP OH P O
OH O O
O
OH CH2OH
O H2C C O P O
OH 1 H2
OH O
Fructose
OH
Fructose−1−P 2
OH
O H O H
HC OH O HC OH Fat
3 synthesis
C O P O C OH
H2 H2
O
Glyceraldehyde−3−P Glyceraldehyde
A deficiency of the brush border enzyme lactase gives rise to a condition named
lactose intolerance, found frequently in people of East Asian descent past their
infant age. If lactose is not cleaved, it cannot be absorbed, so it makes its way
down the drain from the small into the large intestine. Many of the bacteria
found there have the capacity to metabolize lactose, which they will happily
convert to acids and gas. For example, Escherichia coli performs a mixed acid
fermentation. One of the products of this fermentation is formic acid (HCOOH),
which is then cleaved by formic acid lyase to H2 and CO2 .2 This leads to abdom-
inal discomfort and diarrhea. Since the environment in the large intestine lacks
2 The cleavage of formic acid serves the same purpose, namely detoxification of excess acid
Glyceraldehyde
Fructose Fructose-1-P × +
Dihydroxyacetone-P
ADP
ATP
1,3-Bis-P-glycerate
3-P-glycerate
CH2OH
O
CH2OH CH2OH OH
OH O OH O O OH
OH OH OH
OH
OH OH
O
a)
CH2OH HN
O
OH O O O N
CH2OH OH O P O P O C
OH O OH O
O O O
OH
O P OH
OH Glucose 1-P OH OH
O
ADP 2 O
1
ATP CH2OH HN
OH O
CH2OH
OH
O O O N
3
OH O
O P O P O C
OH O
OH
O O
OH
OH
OH OH
O
b)
CH2OH HN
OH O
O O O
OH N
O P O P O C
O
OH
CH2OH O O
O
O
O OH enzyme
OH OH
O P OR + NAD+
OH O
O
CH2OH
probable keto- HN
O
intermediate OH O O O N
OH O P O P O C
OH O
O O
OH OH
Lactose Lactose
Liver
⊗
Galactose Galactose Small
+ + intestine
Glucose Glucose
Lung
bacterium
Liver
Large
CO2 intestine
H2
Figure 4.7 Lactose intolerance. Left: Normally, lactose is hydrolyzed by lactase, and
the constituent sugars absorbed in the small intestine. Right: In lactose intolerance,
lactase is missing, and lactose goes down the pipe into the large intestine, where
bacteria ferment it to hydrogen gas and other metabolites. Exhaled hydrogen can be
used as a diagnostic marker for lactose intolerance.
O OH OH
HC H2C H 2C
HC OH HC OH O
NADPH+H+ NADH+H+
HO CH HO CH HO CH
HC OH HC OH HC OH
HC OH HC OH HC OH
NADP+ NAD+
C OH C OH C OH
H2 H2 H2
Figure 4.8 The polyol pathway. Glucose is reduced to sorbitol by aldose reductase
(left); sorbitol is then dehydrogenated and turned into fructose.
Chapter 5
48
5.1 Pyruvate dehydrogenase 49
Glucose
Glycolysis
Cytosol
Pyruvate
Mitochondrial
transport
Pyruvate
CO2 Pyruvate
Mitochondrion dehydrogenase
Acetyl-CoA
Oxaloacetate Citrate
Citric acid cycle
Malate Isocitrate
H2 CO2
Fumarate α-Ketoglutarate
CO2
Succinate Succinyl-CoA
H 2O
Figure 5.1 The place of pyruvate dehydrogenase and the citric acid cycle in the
complete oxidative degradation of glucose. Glycolysis occurs in the cytosol, whereas
pyruvate dehydrogenase, the citric acid cycle, and the respiratory chain are located
in the mitochondria. A specific transport protein located in the inner mitochondrial
membrane is required to transport pyruvate to the mitochondrial matrix.
This reaction does not occur all at once but instead comprises a sequence
of group transfers and redox steps. We can distinguish five steps altogether,
which involve an equal number of coenzymes. The five reaction steps occur
at three different active sites, which are located on three different types of
enzyme subunits, which are bundled together into one large functional complex.
Pyruvate dehydrogenase therefore is a multi-enzyme complex. In each complex,
there are multiple copies of each of the three individual enzymes. The number
of subunits totals sixty in the E.coli version of pyruvate dehydrogenase (Figure
5.2) and is even higher in mammalian enzymes. Nevertheless, within this large
assembly, each subunit of any type is within easy reach of ones of the other
two types. This proximity ensures an easy flow of intermediate substrates from
one active site to the next during the sequential stages of the reaction, which
greatly increases the overall throughput. High throughput is a key advantage
of multi-enzyme complexes in general.
50 5 Pyruvate dehydrogenase and the citric acid cycle
a)
b)
TPP E1
S S
H
N
Lipoamide O
E2
FAD E3
The types of subunits are named according to the specific partial reactions
they catalyze. The first subunit is called pyruvate dehydrogenase, which name
therefore is ambiguous, denoting both the entire complex and a subunit. The
second subunit is called dihydrolipoyl transacetylase, and the third one dihy-
drolipoyl dehydrogenase. Instead of these explicit names, we will mostly use a
common shorthand notation and call them E1 , E2 and E3 in the following.
Figure 5.3 gives an overview of the sequential steps of the pyruvate dehy-
drogenase reaction:
1. Pyruvate is decarboxylated, and the remaining hydroxyethyl group be-
5.1 Pyruvate dehydrogenase 51
2
CoA SH
HS
E2
H3C S
O
3
S
CoA S CH3 E2
S
O
HS
E2
HS
E3-FAD E3-FADH2
NADH + H+ NAD+
FAD is a coenzyme, since it is in the same state before and after the reaction. NADH is a
cosubstrate, since it undergoes a net turnover, just like the substrate does.
5.3 Regulation of pyruvate dehydrogenase 53
CH3
a) O O b)
+ O P O P OH
N N
O O
S
H3C N NH2 O
CH3
O
H
+ R
N N
S
c) CH3 CH3 H3C N N H H
H
R2 + R1 R2 + R1
N N
C S S
O O
CH3 C CH3
O O O H
O O
+
H
CH3
O
CH3 CH3
H + R
CO2 N N
R2 + R1 R2 R1
N N S
H3C N N H
S O S
H +
H
S C CH3 C C CH3
Lip O
O H O H
S
+
H
CH3 CH3
O
R2 + R1 R2 + R1
N N CH3
OH
S C S
+ R
N N
S C CH3 S C CH3
Lip Lip C S
O H O + H3C N NH
SH SH H H
a) CH3 H
O b) CoA S H
+
C C C C N C C
H
O NH H3C C S
CH3 OH O
O P O C O Lip
O
O NH2 C S C CH3 HS
O P O N
N
O CoA S H
+
C N
O N
H3C C S
O Lip
HS
O OH
O P O
CoA S
O
H3C C HS
O Lip
HS
PDH-P (inactive)
Ca++
Pyruvate +
- NAD+ Phosphatase
Kinase
CoA-SH
+
PDH (active)
- +
Fructose-1,6-bis-P
NADH
Acetyl-CoA
Figure 5.6 Regulation of pyruvate dehydrogenase. Plus signs indicate allosteric acti-
vation, minus signs allosteric inhibition. PDH-P: Phosphorylated pyruvate dehydroge-
nase.
5.4 The citric acid cycle 55
genase complex.
All of the above regulatory effects make good physiological sense. NADH
and acetyl-CoA inhibit PDH, which means that the enzyme will slow down when
its products accumulate. Such feedback inhibition is a straightforward way to
link the activity of a pathway to the metabolic requirements it serves. On the
other hand, pyruvate, NAD+ and fructose-1,6-bisphosphate apply feed-forward
activation – as more substrate arrives, the enzyme should pick up speed.
One more interesting detail is that the PDH phosphatase is activated by
calcium ions. Calcium ions are also involved in triggering the contraction of
muscle cells; activation of PDH will bolster the replenishment of ATP that is
consumed in the contraction.
If we look back at figure Figure 5.1, we see that the TCA produces 4 molecules
of H2 and two molecules of CO2 . Now, if we attempt to balance the acetate with
these amounts of CO2 and H2 :
we see that we are short 4 hydrogens and 2 oxygens on the left side. However,
we can balance the equation if we add two molecules of water:
Therefore, half of the hydrogen produced in the TCA is gained by the reduction
of water. The water-derived oxygen is used to complete the oxidation of the
acetyl carbon. Hydrogen derived both from water and the acetyl group is then
re-oxidized in the respiratory chain to generate ATP.
The energy yield of the TCA itself, in terms of directly generated energy-rich
phosphoanhydride bonds, is very modest – just one molecule of GTP, which
2 The hydrolysis of coenzyme A actually takes place at the stage of citryl-CoA, not acetyl-CoA;
CoA
H2O CoA
SH
O S
+
C O +
C O
BH
H3 BH
H3
CH2 CH2
H2O H2O
a) H2C COOH H2C COOH H2C COOH
c) CO2 NADH+H+
H2C COOH H2C COOH
NAD+
CH2 CH2
O C COOH O C S CoA
Coenzyme A -SH
d)
H2C COOH H2C COOH H2C COOH
O C S CoA O C O P O O C O
O
Phosphate GDP GTP
e)
NADH+H+
NAD+
COOH COOH COOH COOH
FADH2
CH2
FAD HC HO CH C O
COOH 1 COOH
2 COOH 3 COOH
H2 O
Figure 5.8 Further reactions in the citric acid cycle. a: The citrate isomerase reaction
proceeds via a dehydrated intermediate (called cis-aconitate). b: Isocitrate dehydro-
genase transfers hydrogen to NAD+ to generate oxalosuccinate as an intermediate,
which it then decarboxylates. c: The α-ketoglutarate dehydrogenase reaction is anal-
ogous to the one catalyzed by pyruvate dehydrogenase. d: The succinate thiokinase
reaction utilizes the energy-rich thioester bond of succinyl-CoA to generate GTP. The
reaction proceeds via a succinylphosphate intermediate. e: Succinate dehydrogenase
(1), fumarase (2), and malate dehydrogenase (3) regenerate oxaloacetate to close the
cycle.
58 5 Pyruvate dehydrogenase and the citric acid cycle
There are two isozymes of isocitrate dehydrogenase; one uses NAD+ and the
other NADP+ as the cosubstrate. The role of these two isozymes is considered
in section 6.6.
4. α-Ketoglutarate is converted to succinyl-CoA by α-ketoglutarate dehy-
drogenase (Figure 5.8c). This step is completely analogous to the pyruvate
dehydrogenase reaction. The analogy is reflected in a high degree of homology
between the subunits of the two enzymes. If you look closely at the mecha-
nism (Figure 5.3, above), you will see that the reactions carried out by the final
subunit (E3 ) will be identical in both cases, since E3 interacts with lipoamide
when only hydrogen is left but the rest of the substrate is gone. Indeed, the two
enzyme complexes share the very same E3 protein; only E1 and E2 are specific
for the respective substrates. The same E3 occurs yet again in an analogous
enzyme that participates in the degradation of the branched chain amino acids
(section 12.5.2).
5. Succinyl-CoA is converted to succinate by succinate thiokinase, and GDP
is concomitantly phosphorylated to GTP. We know from the glyceraldehyde-3-
phosphate dehydrogenase reaction (Figure 3.6) that thioester bonds are energy-
rich and can drive the phosphorylation of carboxylic acids.3 A carboxylic acid
phosphate, succinylphosphate, also occurs as an intermediate in the succinate
thiokinase reaction (Figure 5.8d). As with phosphoglycerate kinase, the phos-
phate group is then transferred to a nucleotide diphosphate. This nucleotide
is GDP instead of ADP; however, the energy content of the phosphoanhydride
bonds created in GTP and ATP is virtually the same.
6. Succinate is dehydrogenated across the CH–CH bond by succinate de-
hydrogenase to yield fumarate (Figure 5.8e, left). The coenzyme used in this
reaction is flavin adenine dinucleotide (FAD). As a rule of thumb, you can as-
sume that FAD is used in the dehydrogenation of CH–CH bonds, whereas either
NAD+ or NADP+ are used in the dehydrogenation of CH–OH bonds. While all
other enzymes in the TCA are in aqueous solution in the mitochondrial matrix,
succinate dehydrogenase is bound to the inner surface of the inner mitochon-
drial membrane; it is identical with complex II of the respiratory chain (see
section 6.1).
7. Fumarate is hydrated to L-malate by fumarase (Figure 5.8e, middle).
8. Malate is dehydrogenated by malate dehydrogenase to yield oxaloacetate
(Figure 5.8e, right).
The malate dehydrogenase reaction regenerates oxaloacetate, and thus
closes the cycle. It is noteworthy that its equilibrium favours malate, so that
the concentration of oxaloacetate is very low. This has two consequences:
1. The availability of oxaloacetate is a kinetic bottle neck that controls the
rate of the initial reaction of the TCA, that is the synthesis of citrate.
3 With glyceraldehyde-3-phosphate dehydrogenase, the reaction proceeds in the opposite direc-
tion in glycolysis, but it is reversible and functions in the opposite reaction in gluconeogenesis.
5.6 Regulation of the citric acid cycle 59
2. The low concentration of oxaloacetate also detracts from the free energy
that is released by the citrate synthase reaction. To make that reaction go
forward, it is necessary to sacrifice the energy-rich bond of the CoA thioester,
which in contrast to the succinate thiokinase reaction is simply hydrolyzed and
not utilized for the generation of a molecule of GTP or ATP.
In the respiratory chain, the NADH and FADH2 accumulated in the preceding
degradative pathways, mostly the TCA, is finally disposed of by reacting it
with molecular oxygen. The free energy of this “cold combustion” is used
to generate ATP. While a small amount of ATP (or GTP) is produced in those
preceding pathways, the contribution of the respiratory chain is the largest
by far. This is the reason that only aerobic metabolism allows us to sustain
physical exertion for extended periods of time.
The workings of the respiratory chain are quite different from all other
pathways in human metablism (see Figure 6.1). Those other pathways consist of
a succession of discrete enzymatic reactions. In all the ATP-producing reactions
that we have seen so far, the energy was always passed from one energy-rich
bond to the next. In contrast, the respiratory chain combines chemical reactions
with physical forces that are not pinned down to individual molecules, and the
energy is stored and converted in novel ways. The entire process consists of
the following stages:
1. Abstraction of the hydrogen from NADH and FADH2 , and separation of
the hydrogen into protons and electrons.
2. Passage of the electrons down the electron transport chain, which is a
cascade of redox cofactors that are bound to four large protein complexes em-
bedded in the inner mitochondrial membrane. The protein complexes function
as proton pumps; migration of the electrons along this chain makes them expel
protons from the mitochondrion. For each electron migrating down the chain,
multiple protons are pumped out of the mitochondrion.
60
6.1 ATP synthesis can be separated from electron transport 61
Cytosol
H+ H+ H+ H+ H+ H+
+ +
H H
C
I
Q ATP synthase
II III IV
H+ e- e- H+ H+ ADP + P
Mitochondrial matrix
H2O H+
Figure 6.1 Overview of the respiratory chain. Hydrogen provided by NAD+ or FAD is
split into protons and electrons. The electrons are passed along a cascade of proteins
located in the inner mitochondrial membrane, complexes I–IV, that use the energy
provided by the electron flow to pump protons out of the mitochondrial matrix, into
the cytosol. The electrons then react with oxygen and are rejoined by protons to yield
water. The protons accumulated outside the mitochondrion flow back in through ATP
synthase, which is thereby induced to rotate. The rotary motion is then harnessed to
drive the synthesis of ATP from ADP and ionic phosphate. Q: Ubiquinone (coenzyme
Q); C: Cytochrome C. Note that the reactions as shown here are not stoichiometrically
balanced.
3. At the end of the electron transport chain, the electrons are scooped up
by oxygen, which then combines with protons to yield water.
If you think that all this sounds rather strange and vague, you are right –
but don’t let that trouble you. The purpose here is only to divide and conquer,
that is to break up the overall process into manageable parts, which we can
then tackle in more detail in their turn.
62 6 The respiratory chain
a)
O O
H+ NO2 NO2
H+
H+
NO2 NO2
H+
H+
H+
OH
OH OH
OH H+
NO2 NO2
H+
NO2 NO2
b)
H+ H+ H+
C
I
Q
II III IV
- e-
+e
H H+ H+
Figure 6.2 Uncoupling of the respiratory chain. a: The decoupling compound dini-
trophenol can diffuse across the inner mitochondrial membrane in both its protonated
and unprotonated form. It can therefore carry protons into the mitochondrion, thereby
dissipating the driving force for the ATP synthase. In the presence of dinitrophenol,
electron transport and hydrogen oxidation (molecular respiration) will continue, but
ATP synthesis will cease. b: The same thing happens with uncoupling proteins.
can cross the mitochondrial membrane not only in protonated, neutral but also in the deproto-
nated, charged form. This is because the charge in dinitrophenol is highly delocalized, that is
spread across the enrtire molecule.
6.1 ATP synthesis can be separated from electron transport 63
Light H+
H+ H+ ADP+Pi
H+
H+ H+
H+ H+
H+ ATP
H+
from functioning (Figure 6.2a). The same functional effect is brought about by
uncoupling proteins (Figure 6.2b). These are passive proton transporters, lo-
cated in the inner mitochondrial membrane and found in particularly high
concentration in a special tissue called brown fat. Their physiological signifi-
cance is discussed in section 10.3.1.
The effect of uncouplers shows that electron transport can occur in the
absence of ATP synthesis. On the other hand, ATP synthesis will occur in the
absence of electron transport if another means is provided to sustain a proton
gradient. An experimental system, devised by Ephraim Racker, is shown in
Figure 6.3. Here, a molecule of ATP synthase has been incorporated inside
out into a liposome, that is a small artificial membrane particle, along with a
molecule of bacteriorhodopsin. The latter is a quite remarkable protein, found
in a certain bacterium (Halobacterium halobium), that functions as a light-
driven proton pump. Accumulation of protons inside the liposome will set the
ATP synthase in motion and drive the synthesis of ATP.
The significance of these findings is that, despite their presence in the same
membrane and their close proximity, the proton gradient is the only functional
link between the electron transport chain and ATP synthase: The electron
transport chain generates the proton gradient, whereas ATP synthase puts it to
work and thereby dissipates it. Because of this clear separation, we can safely
examine these two functions separately from each other.
64
complex I mitochondrial matrix
complex II
complex III
complex IV
TM domain
Figure 6.4 Structures of the four respiratory chain complexes that form the respiratory chain. The structure of Complex I
is only known in part, and a lame outline has been substituted for the missing trans-membrane domain. Redox cofactors are
highlighted. Yellow/grey blobs represent iron sulfur clusters. Organic rings (green) with grey balls (iron atoms) in the center
are hemes; other rings are flavins or ubiquinone. Drawn with pymol from pdb files 3ias, 2fbw, 3cx5 and 2zxw, after a figure in
Biochemistry 42:2266–2274 (2003).
6.2 The electron transport chain 65
Electrons do not occur in free form but are always part of molecules or ions2
Therefore, to make electrons flow along the prescribed path along complexes
I-IV, these proteins must provide functional groups that are able of accepting
and donating electrons. These groups must be closely spaced, within a few
Angstroms of each other, to allow for efficient electron transfer. Furthermore,
to persuade the electrons to flow in the right direction, the successive transi-
tions must be exergonic, that is their free energy (∆G) must be negative.
In Figure 6.4, you can see a multitude of redox cofactors, neatly spaced along
the protein molecules, that function as “stepping stones” for the migrating
electrons. These prosthetic groups fall into five classes:
2 Electrons do occur free as β-particles in ionizing β-radiation. However, to escape capture,
β-particles must possess an amount of energy much higher than those available in biochemical
or other chemical reactions. This high energy causes them to break up any molecules in their
path into ions or radicals.
66 6 The respiratory chain
O
a)
O P O R
OH O
R = H: FMN
OH R = AMP: FAD
OH
OH
H
H3C N N O
H3C N N O
NH
NH
H3C N
H3C N H
b) R R c)
O
R R
N N H3C CH3O
N N H
R R CH3O
CH3 O
n
R R
d)
Cys Cys Cys
Cys
Cys Cys
Cys Cys
Cys
Cys Cys Cys
Figure 6.5 Structural families of redox cofactors in the respiratory chain. a: Flavine
nucleotides (FAD and FMN), oxidized (left) and reduced (right). b: A heme cofactor with
an iron atom coordinated by the four nitrogen atoms of the ring. The nature of the ‘R’
residues differs in the various heme cofactors, which will contribute to their distinct
redox potentials. c: Ubiquinone. The isoprenyl tail is very long (n=10) and hydrophobic,
so that ubiquinone is confined to the membrane interior. See Figure 6.8 for details on
the redox chemistry. d: Iron sulfur clusters. 1, 2 or 4 of the sulfurs coordinated to
each iron atom can be part of cysteine side chains; the others will be free sulfide.
6.2 The electron transport chain 67
It was stated above that the free energy of the transfer of an electron from one
cofactor to the next must be negative in order to make it happen. Another way
of saying the same thing is that the cofactors should have progressively higher
redox potentials. The reason why redox potentials are commonly used in this
context is that they can be measured more directly than ∆G. However, free
68 6 The respiratory chain
0 0
a) ∆E0 > 0 b) ∆E0 < 0
e–
e– e–
e– e–
e– e– 2 e– 2 e–
1
2 H2 H+ Q Q+ 2 H+ H2 NADH NAD+
+ H+
Figure 6.6 Experimental setup for measuring standard potentials. The sample and
the reference are contained in two adjacent chambers. Platinum electrodes are im-
mersed in the solutions. Electrons are withdrawn from one chamber and delivered into
the other, flowing through a voltmeter indication the direction and magnitude of the
potential difference. Protons and other ions can flow across a salt bridge to maintain
electroneutrality. Left: Coenzyme Q withdraws electrons from the standard hydrogen
0
electrode and accordingly has a positive ∆E0 . Right: NADH feeds electrons into the
0
standard electrode, making its ∆E0 positive.
energy and redox potential are directly related by the following equation:
∆G = −∆E n F (6.1)
3 Another thing to remember is that the unit of ∆E, Volt, is defined as J/Coulomb, since voltage
= energy/charge.
The minus sign in this equation results from the fact that the electron-donating electrode, the
cathode, is considered negative by convention. It is fundamentally meaningless but handy as a
trap in exam questions.
Faradays constant can be considered a historic artifact, resulting from the fact that units of
electricity were already defined before the principles of electrochemistry were understood.
4 You have encountered the same concept with chemical elements as their electronegativity:
An element with a high electronegativity holds on to electrons particularly firmly, that is it has a
high affinity for electrons.
6.2 The electron transport chain 69
complex III
Heme c1 CytC
∆E0 (V)
0.2 −40.6
Heme a3
0
0.4 −79.2
O2
0.8 −156.4
1.0 −195.0
Figure 6.7 Redox potentials for selected cofactors along the respiratory chain. Elec-
trons are fed either from NADH into complex I or from FADH2 into complex II. The
scale on right hand side shows the conversion of ∆E to the molar ∆G, per pair of elec-
trons (see equation 6.1). As ∆E goes up, ∆G goes down, driving the electron transport
forward. Major jumps in potential occur within complexes I, III and IV; these are the
sites that harness the free energy of electron transport to proton export. Abbreviations:
CoQ, coenzyme Q (ubiquinone); CytC, cytochrome C; Fe-S, iron sulfur cluster.
1. NADH, FADH2 , FMNH2 and coenzyme Q carry both electrons and protons
– that is, hydrogen. In contrast, the hemes and the iron-sulfur clusters carry
only electrons.
2. NAD+ can only accept and donate pairs of electrons, whereas the hemes
and iron-sulfur clusters can only accept and donate single electrons.
The switch from the two-electron carrier NADH to the one-electron carrying
Fe−S clusters within complex I is mediated by FMN, which can accept or donate
electrons both pairwise and singly:
- NAD+ + FMNH2
NADH + H+ + FMN ------→ (6.2)
III · + II
FMNH2 + Fe −S ------→
- FMNH + H + Fe −S (6.3)
· III + II
FMNH + Fe −S ------→
- FMN + H + Fe −S (6.4)
Fe II −S + H+ + Q ------→
- QH + Fe III −S
·
(6.5)
Fe II −S + H+ + QH ------→
·
- QH2 + Fe III −S (6.6)
a) e-
O O
CH3O CH3 CH3O C CH3
H H
CH3O CH3O
O CH3 n O CH3 n
OH O
CH3O
2H+, e-
CH3 CH3O CH3
H H
CH3O CH3O
OH CH3 n O CH3 n
b) 2 H+
CytC
QH2 QH2 Q e-
Q Q-
2 H+
CytC
Q e- QH2
QH2 Q-
2 H+
Figure 6.8 The ubiquinone cycle, criminally simplified. a: Redox chemistry of
ubiquinone (coenzyme Q). It can accept two electrons successively and two protons
successively. b: The ubiquinone cycle (simplified so that I can understand it). Complex
III has two binding sites for ubiquinone . Oxidation of one molecule of ubiquinone
partially reduces the second one; oxidation of a new molecule of ubiquinone com-
pletes reduction of the second one and causes uptake of two protons from the cytosol.
Protons released by oxidation are at all times released at the cytosolic side.
6.2 The electron transport chain 73
As pointed out above, some of the protons that are being expelled from the
mitochondrion are accepted from the hydrogen carriers NADH and ubiquinone
and travel together with electrons for a part of the journey. However, at some
point they must part company, and the protons must be expelled, whereas
the electrons are retained. Also, more protons are being expelled than can be
accounted for by the hydrogen carriers. In particular, complex IV does not
interact with any hydrogen carriers yet expels up to four protons for each pair
of electrons accepted. So, there must be mechanisms that extract energy from
the transfer of proton-less electrons and apply it towards the expulsion of
electron-less protons. How does this work?
The experimental evidence on this point is pretty fragmentary, and to the
extent they are understood the emerging mechanisms are quite complex. There-
fore, instead of trying to describe them faithfully, I will present a simplified
conceptual model to provide an idea of how things work (Figure 6.10).
The basic idea is that binding and release of electrons cause conformational
changes to a protein. This is entirely analogous to conformational changes
caused by allosteric effectors binding to enzymes, or to phosphate groups
bound to proteins (e.g. myosin light chain). An electron carries a charge, a
charge causes a field, and a field will cause forces that act on charged residues
on the protein, so it is quite easy to see how migrating electrons can cause
conformational changes.
5 The toxicity of partially reduced oxygen species such as O2– is actually exploited by phago-
cytes, which release them intra- and extracellularly to destroy microbes. Enzyme defects in the
production of these reactive oxygen species produce severe immune deficiencies.
74 6 The respiratory chain
a) R R
R R
N N
N N
R R
R R
O
O
His His
His
b)
2+ 3+ 4+ iron
e-
O O- O 2-
O O- O2- H+
H+
1+ 2+ 2+ copper
e-
H+
O2
2+ 3+ 3+ iron
2 e- 2 H+
O2- H+
2 H 2O O2- H+
1+ 2+ 2+ copper
+
mitochondrial
matrix + – +
+ +
cytosol
–
–
+
Figure 6.10 A conceptual model linking electron transport to proton pumping. The
proton pump sits between two other elements of the electron transport chain. It
possesses three electron carrier cofactors (white circles). In the resting state, the
proton binding site is open to the mitochondrial matrix. Arrival of an electron at the
first carrier causes a conformational change that everts the proton binding site, so that
it now communicates with the cytosol and ejects the proton, and at the same time
facilitates migration of the electron to the next cofactor. The pump then reverts to its
resting position.
It is commonly stated that about 10 protons are ejected for each pair of elec-
trons abstracted from NADH, such that 4 protons are ejected at each of com-
plexes I and IV, and 2 at complex III.6 Complex II does not eject any protons
but just abstracts them from FADH2 and passes them on to ubiquinone. If you
look at Figure 6.7, you will notice that the difference in the redox potentials of
FAD and ubiquinone is rather small. Consequently, the amount of free energy
associated with the transfer of electrons from FAD to ubiquinone is too small
to permit the performance of work against the proton gradient.
6 Note that this number is at variance with the model of the coenzyme Q cycle given above.
Generally speaking, the figures given for the numbers of protons ejected at each stage vary quite
a bit between various texts.
76 6 The respiratory chain
The proton concentration in the cytosol is approximately ten times higher than
that in the mitochondrial matrix. How much free energy does it generate if
protons travel downhill this concentration gradient? This can be determined
from the following formula:
[H + ]in
∆G = RT ln (6.7)
[H + ]out
+
With R = 8.31 J/K mol, T = 310K, and [Hin ]/[Hout
+
] = 10 this comes to roughly
6 kJ/mol. While this is significant, the larger contribution to the proton-motive
force comes from the electrostatic membrane potential across the inner mito-
chondrial membrane. Like the proton concentration gradient, this electrical
potential is a direct consequence of the proton pumping: Each proton ejected
leaves a deficit of a positive charge, or one negative charge, inside the mito-
chondrion. In a fully energized mitochondrion, the resulting potential amounts
to 150 mV, negative inside. The free energy that this membrane potential con-
fers to each proton can again be calculated from equation 6.1 and works out to
approximately 15 kJ/mol. In summary, the proton-motive force is caused roughly
to 3/4 by the membrane potential, and to 1/4 by the proton concentration gradi-
ent.
c)
P
P
AD
ATP
ATP
3. The β-subunit, which contains the active site of the enzyme, utilizes the
energy transmitted by these conformational changes to synthesize ATP (Figure
6.11c).
How does the β-subunit transform the energy of rotation into chemical
energy for ATP synthesis? Strictly speaking, it doesn’t – it turns out that the
isolated β-subunit can create ATP all by itself. In doing so, the β-subunit adopts
distinct conformational states. In the first state, it binds ADP and phosphate.
Once both are bound, β transitions to the next state, which binds ATP with
exceptionally high affinity but no longer binds ADP and phosphate. Formation
of the several non-covalent bonds between β and ATP provides the energy that
78 6 The respiratory chain
Figure 6.12 A hypothetical model for coupling of proton flux and ATP synthase
rotation. See text for details. Source: P. Boyer, Nature 402:247–249 (1999).
instead of just oscillating? This would save it the trouble of working while
still permitting proton flux. A steam engine gets around a similar problem by
inertia, which is created by adding a nice, heavy, cast-iron flywheel. That’s not
possible here, because of the minuscule dimensions.8 Accordingly, there must
be a tighter coupling of proton flux and rotation that most likely involves some
conformational flexibility in both the a and the c subunits. There are some
experimental data to support this assumption.
There is, however, one interesting finding that the model in Figure 6.12 does
account for: The number of protons transported per rotation is identical to
that of the c subunits in F0 . Intriguingly, ATP synthases in different organisms
vary in their number of c subunits. This will directly affect the stoichiometry of
ATP synthesis and proton transport. It would be interesting to know whether
there are complementary variations in the number of protons driven out per
electron during electron transport. If not, the different subunit stoichiometry
of F0 should directly translate into a different ATP yield in the entire respiratory
chain.9
We can now determine how much ATP is produced through the complete oxi-
dation of glucose via glycolysis, TCA, and respiratory chain. Here are the bits
and pieces:
1. For each molecule of glucose, a total of 10 moles of NADH and 2 FADH2
are produced by glycolysis, pyruvate dehydrogenase, and citric acid cycle.
2. 10 protons are exported per NADH, and 6 per FADH2 in the respiratory
chain, for a total of 112 protons.
3. Each revolution of ATP synthase consumes 10 protons and generates 3
ATP.
Overall, we obtain 112 protons per glucose × 3 ATP per 10 protons, or
33.6 ATP per molecule of glucose. Therefore, 33.6 ATP could be generated
in the respiratory chain for each molecule of glucose degraded, if all protons
were available for driving ATP synthase. However, some protons are diverted
to other purposes, so that the actual yield will be lower than this theoretical
value. Most importantly, some protons are needed for ATP transport. ATP
synthesized in the mitochondrion needs to be exported to the cytosol, and
ADP produced there needs to get back in. This is accomplished by a special
8 A student who took this class in 2005, Kelvin Cheung, took up the challenge to calculate the
kinetic energy of rotating ATP synthase; it works out to about one billionth of the energy required
for making 1 ATP. Honorable mention.
9 If you are interested in this problem, you are welcome to dig up and some recent information
on this and discuss it with me. If it merits inclusion here, it will earn you an honorable mention
and a recommendation letter.
80 6 The respiratory chain
component but not the proton concentration component of the proton- motive force that gets
dissipated. Then, it costs about 3/4 of the energy of a pumped proton to export one ATP.
6.5 Shuttle systems for the NADH re-oxidation 81
c)
IV
C
III
Glycerol-P Glycerol-P
Q
NAD+ 2 e-
GPD
(FAD)
NADH+H+
II
DHAP DHAP
I
2 H+
might go at full blast even when the demand for ATP is low and NADH is
high! How, then, is this enzyme prevented from uncontrolled consumption of
isocitrate?
It appears that, at least during times of low demand for ATP, NADP+ -
dependent isocitrate dehydrogenase is close to equilibrium. This equilibrium is
sustained by a high mitochondrial level of NADPH, which in turn is maintained
by NAD+ /NADPH transhydrogenase. This remarkable protein, wich is located
in the inner mitochondrial membrane, is both an enzyme and a transporter. It
reduces NADP+ to NADPH at the expense of NADH. As with ATP synthase, the
enzyme reaction is coupled to the translocation of protons:
NADH + NADP+ + H+ + +
out → NAD + NADPH + Hin (6.8)
11 This is my take on the subject. There is, however, considerable variety of opinion on the role
mitochondrial
matrix
NADPH NAD+
H+
H+ cytosol
NADPH NAD+
H+
H+
Figure 6.14 Function of NADP+ /NADH transhydrogenase, and its integration with the
TCA and the respiratory chain. a: Function of transhydrogenase in idling mode, while
the demand for ATP is low. In this situation, the extra-mitochondrial H+ concentration
is high. Protons enter through transhydrogenase and sustain a high concentration
of NADPH, some of which in turn is dissipated by NADP+ -dependent isocitrate dehy-
drogenase. b: Function of transhydrogenase at times of high demand for ATP. A high
throughput of the respiratory chain causes the extra-mitochondrial H+ concentration to
drop. The proton flow through transhydrogenase now reverses, as does the operation
of NADP+ -dependent isocitrate dehydrogenase. This results in the provision of extra
NADH for the respiratory chain, as well as protons for ATP synthesis.
Chapter 7
Gluconeogenesis
The metabolic pathways we have considered so far account for the complete
oxidative degradation of glucose. Glucose is the most important substrate of
energy metabolism for several reasons:
1. Glucose accounts for a large share of all calories in a typical human diet.
2. Some cell types, for example erythrocytes, depend entirely on it for all
their metabolic energy. Others, such as nerve cells, only very reluctantly give
up their preference for it, althougy they can adapt to ketone bodies (which
are formed by fatty acid degradation) as alternative substrates in times of
starvation.
3. Glucose is the major source of NADPH, which is needed for many biosyn-
thetic tasks, including fatty acid and sterol synthesis. The pathway that regen-
erates NADPH by oxidation of glucose—the hexose monophosphate shunt—will
be discussed later on.
It is plausible then that a pathway should exist that can turn other sub-
strates into glucose if the latter is lacking in the diet. In fact, many carnivorous
mammals and many humans—and not merely decadent Westerners,think of the
traditional lifestyle of Inuit—live on diets that are very rich in protein but not
glucose. Such diets will typically also be rich in fat; however, fatty acids cannot
be converted to glucose in the mammalian metabolism. Amino acids therefore
are the major source of carbon for the synthesis of glucose. The pathway of
glucose synthesis is called gluconeogenesis.
85
86 7 Gluconeogenesis
Oxaloacetate Citrate
Malate Isocitrate
CO2
Fumarate α-Ketoglutarate
CO2
Succinate Succinyl-CoA
Amino acids
R R
S S
c) O
O
Enzyme-B H3C C C
O
O
O
Enzyme-BH+ H2C C C
O
Biotin-CO2
Biotin
O O O
C C C
H2
O O
The final reaction in glycolysis is the transfer of the phosphate group from
phosphoenolpyruvate (PEP) to ATP. This reaction is irreversible because of the
strongly exergonic nature of the accompanying rearrangement of pyruvate from
the enol to the keto form (see section 3.4.4). In gluconeogenesis, it takes two
enzymatic steps to turn pyruvate back into PEP: (1) Carboxylation to oxaloac-
etate by pyruvate carboxylase (Figure 7.2), and (2) conversion of the latter to
PEP by phosphoenolpyruvate carboxykinase (Figure 7.3a).
Pyruvate carboxylase requires biotin as a coenzyme. This coenzyme is
88 7 Gluconeogenesis
We can combine these two equations and cancel glucose and glucose-6-
phosphate, since they occur on both sides. We obtain:
ATP + H2 O ------→
- ADP + phosphate (7.1)
7.3 Regulation of gluconeogenesis 89
a) O O
CO2
O
C C H2C C
H2
O OH OH
O O
O
N
NH
O O O HC
O
H2
H2C C O P O P O P O C N
O N NH2
OH
O O O O HC CH
O P O HC CH
OH OH OH
b) Fructose-1,6-bis-phosphate
H 2O
1 phosphate
Fructose-6-phosphate
2
Glucose-6-phosphate
H 2O
3
phosphate
Glucose
This means that the simultaneous activity of the two enzymes would cause
unrestricted, idle hydrolysis of ATP. A pathway with no other effect than the
expenditure of ATP (or some other form of metabolic energy) is called a futile
cycle. Futile cycling will also result from simultaneous activity of of fructose-
1,6-bisphosphatase with phosphofructokinase, and of pyruvate kinase with
pyruvate carboxylase and PEP carboxykinase, with the latter differing only by
hydrolysing GTP instead of ATP.
It is clear that unchecked operation of futile cycles would be a disaster – all
ATP would be rapidly consumed, and the energy simply be dissipated as heat.
Of note, the liver has a temperature well above the body temperature (39-40 ◦ C),
and a fair share of the process heat generated in liver metabolism is believed
90 7 Gluconeogenesis
to derive from the futile cycles just discussed. Still, there are regulatory mecha-
nisms to keep these cycles in check. While this applies to all enzymes involved,
as an example we will consider those that concern phosphofructokinase and
fructose-1,6- bisphosphatase.
2 ADP ------→
- ATP + AMP
This allows for a makeshift regeneration of some ATP from ADP. Also, if we
consider the equilibrium:
we see that [AMP] will vary with the square of [ADP]; [AMP] therefore is the
more sensitive parameter to detect changes in the availability of ATP.
Fructose-1,6-bisphosphatase is stimulated by ATP and inhibited by AMP.
This behaviour is opposite to that of phosphofructokinase, and it ensures that
one enzyme will be active when and only when the other one is inactive, so that
futile cycling and ATP hydrolysis is avoided.
a) Pi Fructose-6-P ATP
+ ATP -
Fructose-1,6 Phospho-
bisphosphatase - AMP + fructokinase
- Fructose-2,6-bis-P +
H 2O Fructose-1,6-bis-P ADP
b) O O O
O P OH O P OH O P OH
O O O OH
CH2 O H2C CH2 O H2C
OH OH
OH O
OH OH HO P O
O
c) Epinephrin,glucagon Insulin
+
+
Protein kinase A
ATP ADP
P
PFK-2/(bis-P’ase) (PFK-2)/bis-P’ase
Fructose-2,6-bis-P
Fructose-6-P
Insulin ↑ → cAMP ↓ →
PFK-2 ↑, fructose-2,6-bisphosphatase ↓ → fructose-2,6-bisphosphate ↑ →
glycolysis favoured
glucose glucose
Figure 7.5 The Cori cycle. Lactate generated from glucose by anaerobic glycolysis in
the skeletal muscle during exercise is moved to the liver, reoxidized to pyruvate and
turned back into glucose by gluconeogenesis, which is then returned to the muscle or
other peripheral tissues.
Glyoxylate
O O
b) Glucose
Acetyl-CoA
Citrate
Oxaloacetate
Isocitrate
Malate
1 Glyoxylate
Fumarate
Acetyl-CoA
Succinate 2
Figure 7.6 The glyoxylate cycle of plants. a: Two enzyme reactions are required in
addition to those of the TCA. Both reactions are similar to the citrate synthase reaction.
b: Overview of the cycle. Dotted arrows represent reactions of the TCA that are skipped
by the glyoxylate cycle. (The conversion of oxaloacetate to glucose has been abridged.)
Glycogen metabolism
O O O O O
CH2
O O O O O O
O O O O O
CH2 CH2
O O O O O O O O-Tyr Glycogenin
3. Chain elongation,
4. Introduction of branch points.
OH OH O P O P O P O C
O UDP-glc UDP-glc UDP-glc UDP-glc UDP-glc
OH O O O
CH2OH
Glucose-6-P O
OH O OH OH
OH O P O
O
HO O O O O O O-Tyr Glycogenin
OH OH
Glucose-1-P CH2OH
HN
O
UDP UDP UDP UDP UDP
OH O O O N
OH O P O P O C
O
2 Pi P-Pi OH OH O
UDPglucose OH OH
b) CH2OH
HN
d)
O CH2OH
OH O O O N
OH O P O P O C
HO Tyr Glycogenin O O O O O O O O-Tyr Glycogenin
O
OH OH O
OH OH
HN
HO O O
O
O O O CH2OH
N
8 Glycogen metabolism
HO P O P O C
O
O CH2
O O OH
OH O HO O O O O O-Tyr Glycogenin
Tyr Glycogenin
OH OH OH
UDP
Figure 8.2 Reactions in glycogen synthesis (1). a: Activation of glucose. Glucose-6-phosphate is converted to glucose-1-phosphate
by phosphoglucomutase. Glucose-1-phosphate uridylyltransferase converts glucose-1-phosphate to UDP-glucose. Pyrophosphate
released is cleaved by pyrophosphatase. b: 1 molecule of UDP- glucose reacts with glycogenin. c: Glycogen synthase linearly
polymerizes glucose residues, again using UDP-glucose as a substrate. d: Branching enzyme transfers the end of a growing chain
onto the C6 atom of an internal glucose residue. Both C4 ends are subsequently extended by glycogen synthase until branching
repeats.
8.2 Glycogen degradation 101
amylose; however, the lysosomal enzyme is a separate entity and has an acidic pH optimum, in
keeping with the acidic environment inside the lysosomes.
102 8 Glycogen metabolism
CH2OH
a) O
CH2OH
OH
O
O Glycogen
O OH
OH
CH2OH
CH2OH O Glycogen
O O
H O OH
OH
OH H
OH O UDP OH
OH O UDP
CH2OH OH
OH
CH2OH O Glycogen
O
O OH
OH
OH H +UDP
OH
CH2OH CH2OH
b)
O CH2OH O
H
OH O OH
+
OH O UDP OH C H O Glycogen
O
OH OH OH
OH
O UDP
CH2OH CH2OH
O H O
OH OH
OH O Glycogen
O
OH OH
a) Pi
glc–glc–glc–glc–glc–glc–glc–glc
glc-1-P
Pi
glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc
glc-1-P
b)
glc–glc–glc–glc
glc–glc–glc–glc–glc–glc–glc–glc–glc–glc
glc
glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc
glc
glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc
Glucose-1-P
+
+
Protein kinase A
Glycogen Glycogen
synthase (active) synthase–P (inactive)
Phosphorylase Phosphorylase
kinase (inactive) kinase–P (active)
Phosphorylase-P Phosphorylase
(active) (inactive)
105
106 9 The hexose monophosphate shunt
Glycogen UDP-Glucose
Fructose-6-phosphate
Pyruvate
6 CO2 + 6H2O
a) Glucose-6-P biosynthesis
of nucleotides and
nucleic acids
Regenerate 5 2 NADP+
Glucose-6-P 2 NADPH
from 6 Ribulose-5-P CO2
Ribulose-5-P Ribose-5-P
b) CO2
OH OH
OH OH O
OH OH 1 OH
OH OH
H 2O
2
O
HO
Ribulose-5-P NADPH+H+
OH OH
NADP+ HO 6-P-Gluconate
O
OH OH
3
OH O OH O
CO2
Nucleotides O P OH O P OH
O O
Figure 9.3 The oxidative stage of the hexose monophosphate shunt. Enzymes:
1: glucose-6- phosphate dehydrogenase; 2: 6-phosphoglucolactonase; 3: 6-
phosphogluconate dehydrogenase.
The reactions in the second phase of the hexose monophosphate shunt are
depicted in Figure 9.4:
1. Two molecules of ribulose-5-phosphate are converted to xylulose-5- phos-
phate and to ribose-5-phosphate by ribulose-5-phosphate epimerase and ribu-
lose-5-phosphate isomerase, respectively.
2. Transketolase transfers a C2 unit from the xylulose-5-phosphate to the
ribose-5-phosphate, yielding glyceraldehyde-3-phosphate and the C7 sugar se-
duheptulose-7-phosphate.
3. Transaldolase transfers a C3 unit from the seduheptulose-7-phosphate
back to glyceraldehyde-3-phosphate, yielding fructose-6-phosphate and the C4
sugar erythrose-4-phosphate. The fructose-6-phosphate may enter glycolysis,
or may be converted back to glucose-6-phosphate by phosphohexose isomerase.
4. Transketolase transfers a C2 unit from another molecule of xylulose-5-
phosphate to erythrose-4-phosphate. This yields a second molecule of fructose-
6-phosphate and again glyceraldehyde-3-phosphate. Both may enter glycolysis
or be converted back to glucose-6-phosphate.
The conversion of glyceraldehyde-3-phosphate back to glucose-6-phosphate
via fructose-1,6-bisphosphate utilizes the enzymes from gluconeogenesis and
glycolysis that you so fondly remember from the previous chapters. Recall
that aldolase needs two molecules of triose phosphate to form one molecule of
fructose-1,6- bisphosphate. This results in an overall stoichiometry of 2.5 mo-
lecules of hexose per 3 molecules of pentose, or five hexoses per six pentoses.
9.1 Reactions in the hexose monophosphate shunt 109
OH
OH OH O
O O HO
OH HO TK O OH
RE
OH OH OH OH
O P O P O P O P
C5 C5 C3 TA C6
OH
O OH
OH O HO O
O OH OH O HO
OH OH OH OH OH
RI
OH OH OH OH OH
O P O P O P O P O P
C5 C5 C7 C4 C6
TK
O P
OH OH O
O O HO
OH HO O OH
RE GN
OH OH OH OH
O P O P O P O P
C5 C5 C3 0.5 C6
The juggling of sugar chain length depicted in Figure 9.4 is brought about
by two enzymes, transaldolase and transketolase. While the sugar substrates
they act upon appear to be quite varied at first glance, they fall into just two
structural classes, the members of each of which differ only in chain length
(Figure 9.5). The two enzymes only interact with the topmost parts of these
sugar molecule, so that the chain length of the remainder doesn’t enter into
the picture and does not create a need for separate enzymes. The basic idea of
chain length variation is that transketolase always transfers two-carbon units,
whereas transaldolase always transfers three-carbon units. Sequential reaction
110 9 The hexose monophosphate shunt
OH OH OH OH
O O O O
OH HO HO HO
OH OH OH OH Ketoses
O P O P OH OH
Ribulose-5-P O P OH
O
O P
OH O
OH OH O
Aldoses
OH OH OH
O P O P O P
of the two enzymes will therefore lead to a net change of the substrate chain
length by one carbon.
therefore can go either way and bring about the interconversion of pentoses,
hexoses and sugars of other lengths as needed.
no difference in their redox potentials, i.e. they have the same standard redox potentials.
3 This ∆G is similar to that provided by the hydrolysis of ATP to ADP. For the cytosol, this
sounds about right (see section 9.4 below). It is probably less in mitochondria, where NADH and
NADPH are distinguished only by the energy of a single proton transfer (via transhydrogenase).
112 9 The hexose monophosphate shunt
R
a)
S H2C OH
H3C
R
+ C O H
N
S H2C OH R HC OH
H3C
+ C
N C O H+ HC OH
R HO CH R HC OH
HC OH
HC OH
S H2C OH
H2C O P H3C
OH C O P
+ C H2
N
R
H2C OH
R +
O
+ H+
H
S H2C OH HC O HO CH
H3C
+ C OH HC OH HC OH
N
R O CH HC O HC OH HC OH
H
HC OH HC OH HC OH HC OH
HC OH HC O H HC O
HC OH HC OH HC OH
HC OH H 2O HC OH
H+ HC OH
HC OH HC OH HC OH
H 2O
H2C O P H2C O P C O P
H2
Liver
Urine
b)
NADP+
O O
H H
C2H5 N NADPH+H+ C2H5 N
O O
N HO N
H H
O O2 H2O O
O
O O H O O
C C2H5 N C
O O O
OH O N OH
H
OH O UDP OH O UDP
OH OH
Figure 9.7 Role of NADPH in drug metabolism. a: Overview: Drug metabolism occurs
in the liver and often serves to convert hydrophobic drug molecules to hydrophilic
metabolites, which facilitates excretion in the urine. b: Example: NADPH- dependent
hydroxylation of the drug phenobarbital. Hydroxylation is followed by conjugation
with glucuronic acid, which is derived from UDP-glucuronic acid.
The enzymes are contained in small vesicles called peroxisomes, which will
fuse with phagosomes that contain ingested microbes. The reactive oxygen
species (ROS) generated have an important role in the destruction of the mi-
crobes. This is shown by the fact that enzyme defects in the formation of ROS
cause severe immunodeficiencies, in particular with respect to bacterial infec-
tions. ROS also have an important share in the tissue destruction that occurs
in inflammation.
9.6 Glucose-6-phosphate dehydrogenase deficiency 115
2 O 2- + 2 H + H 2O 2 + O 2
Superoxide dismutase
b)
ROS
O
a)
O O
N SH
H
NH
O O
O NH2
O O
O O O O
N S S N
H H
NH N
H
O O O O
O NH2 NH2 O
Glutathione Glutathione
peroxidase reductase
c) O
O
HN
Glutathione
spontaneous spontaneous
peroxidase
O
GSSG OH O2 GSSG
HN
H3C N NH2
Figure 9.9 The role of glutathione in scavenging of reactive oxygen species, and
the mechanism of glutathione depletion in favism. a: Glutathione in its reduced and
oxidized states. b: Glutathione peroxidase oxidizes glutathione and reduces hydrogen
peroxide to water. Reduced glutathione is regenerated using NADPH by glutathione
reductase. c: Isouramil is one of several similar compounds found in broad beans.
It cycles between reduced and oxidized states; each cycle oxidizes four molecules of
glutathione.
9.6 Glucose-6-phosphate dehydrogenase deficiency 117
Triacylglycerol metabolism
Various types of lipids occur in the human body: (1) Triacylglycerol, (2) choles-
terol, and (3) polar lipids, which include phospholipids, glycolipids and sphin-
golipids.
While polar lipids and cholesterol are found in the cell membranes of ev-
ery cell, triacylglycerol is essentially confined to fat tissue, which stores and
releases it, and to the cells in the intestine and the liver that synthesize and
degrade it.1 Yet, triacylglycerol is the most abundant lipid species, and the only
one with an important role in energy metabolism. We will therefore here focus
on triacylglycerol. Cholesterol, which is not important in energy metabolism,
will be covered in a separate chapter as well because of its medical importance.
Triacylglycerol occurs in human metabolism in two roles: (1) As a foodstuff.
A significant fraction of our caloric intake is triacylglycerol. (2) As an endoge-
nous store of metabolic energy. This storage can be replenished from dietary
fat, or by endogenous synthesis of fat from carbohydrates or proteins.
The amount of energy per gram of tissue is far higher in fat than in any
other tissue, for two reasons: (1) One gram of triacylglycerol itself contains
roughly twice as many calories as one gram of carbohydrates or protein. This is
because triacylglycerol contains much less oxygen than carbohydrates, in which
oxygen contributes half the mass but essentially no metabolic energy. Similarly,
1 There is also some intracellular storage of triacylglycerol, for example in the muscle cells,
118
Triacylglycerol metabolism 119
H2 H H2 OH
a) C C C
O O O
OH OH OH O
O O O CH2
Glycerol H2C
CH2
H2C
CH2
H2C
CH2
H2C
CH2
H2C
CH2
H2C
CH2
Palmitic acid
H2C
(hexadecanoic acid)
CH3
b) ketone bodies
5 6 ADP ATP
3
fatty acids acetyl-CoA CO2 + H2O
4 7
1 2
Triacyl-
glycerol
glycerol
glucose pyruvate
protein contains oxygen, nitrogen and sulfur and is similar in energy content
per dry weight to carbohydrates. (2) Triacylglycerol in fat cells coalesces to
droplets that are entirely free of water. In contrast, protein and carbohydrates,
including glycogen, always remain hydrated.
Because of this efficiency of storage, it makes sense that most of the excess
glucose or protein is converted to fat, while only a limited fraction is stored as
glycogen. There is, however, one limitation to the usefulness of triacylglycerol:
Once carbohydrate or protein carbon has been converted to fat, it can go back
and forth between fat, fatty acids, acetyl-CoA, and ketone bodies, but it is no
longer available for the regeneration of glucose (Figure 10.1b). Therefore, when
starving, we will always have to degrade some protein along with fat to keep
up a minimum supply of glucose, and therefore starving people from day zero
will not only deplete their fat stores but also their muscle tissue.
a)
Fat
Monomeric
detergent
b) c)
OH O OH
O O
O
micellar
CMC
monomer
3 Sooner or later, each particle in the blood will pay the liver a visit, even if it was initially
bypassed.
4 Interestingly, the surfaces of the intestinal cells withstand high concentrations of bile and
fatty acids! This phenomenon has not yet received appropriate scientific attention.
10.1 Utilization of dietary triacylglycerol 123
Chylomicrons
Chylomicrons lymphatics
b)
Fat cell Triacylglycerol
Endothelial
cell
Fatty acids,
glycerol
Chylomicron
to Liver
Lipoprotein lipase
Figure 10.3 Intestinal uptake and post-processing of dietary fat. a: Left: Fatty acids
and monoacylglycerol are taken up by the intestinal cells, converted back to triacyl-
glycerol, and together with apolipoproteins packaged into chylomicrons. Right: Chy-
lomicrons (and other lipoproteins) are fat droplets surrounded by a protein shell. b:
Lipoprotein lipase binds chylomicrons and extracts and cleaves triacylglycerol. The
cleavage products are stored in fat cells (after being converted to triacylglycerol yet
again, as shown) or utilized by oxidative degradation.
124 10 Triacylglycerol metabolism
a) CH3
+
H3C N CH3
O
CH2
Acyl-carnitine HC O
CH2
COOH
Acyl-CoA CoA S
b) OM IM
ADP
Fatty acid
CoA Carnitine Carnitine CoA
CoA
Acyl-carnitine Acyl-carnitine
I hope that you noticed the similarities of the enzyme reactions discussed
above to some others we have seen before. At least in the first three steps,
the similarity to reactions in the citric acid cycle—succinate dehydrogenase,
fumarase, and malate dehydrogenase—are quite straightforward. Also notice
the consistent use of redox coenzymes (NAD+ and FAD) in the two pathways:
1. Whereever a CH−OH bond is dehydrogenated, NAD+ or NADP+ is employed
126 10 Triacylglycerol metabolism
a) O β
CoA S ω
α γ FAD
O 1
FADH2
CoA S
O OH 2
CoA S
NAD+
O O 3
NADH++H+
CoA S
CoA-SH
4
O
β
CoA S CH3 O
CoA S
O 1
CoA S
O
b) O
CoA S
CoA S CH3
Acetyl-CoA
O O
CH3
CoA S CoA S C
Propionyl-CoA
5 This also holds in other pathways and for CH−NH bonds. The only exception I’m aware of is
O O
CoA S
+
BH
Enzyme S B
+
H
Enzyme S
O
O
CoA S
CoA S
O
B
Enzyme S
CoA S
Figure 10.6 Mechanism of the thiolase reaction. See text for details.
FAD is employed.
As to the thiolase reaction, we have not seen a completely analogous one.
However, if we look at the individual steps of this reaction, we can still recog-
nize some familiar features (Figure 10.6):
1. The nucleophilic attack of a cysteine sulfur in the active site on a carbonyl
group in the substrate yields a covalent intermediate. This also happens with
glyceraldehyde-3-phosphate dehydrogenase.
2. Acid-base catalysis breaks a carbon-carbon bond adjacent to the carbonyl
bond. Note that the thiolase reaction is reversible. Making of a carbon-carbon
bond adjacent to the carbonyl group by acid-base catalysis then occurs in the
reverse reaction, and we have seen that before with citrate synthase and malate
synthase.
3. The creation of one thioester at the expense of another occurs in the
second step of the pyruvate dehydrogenase reaction.
Propionyl-CoA Succinyl-CoA
O O
CH3 CH2
CoA S CH2 CoA S CH2 COOH
ATP
CO2
1
3
ADP
O O
H3C 2 H3C
C S CoA C S CoA
COOH COOH
(S)-Methylmalonyl-CoA (R)-Methylmalonyl-CoA
TAG
glycerol
+
FA FA acetyl-CoA
ketone
ketogenesis bodies
acetyl-CoA
You know from experience that fat tissue is mostly white, which is due to
the fact that typical fat cells contain little else than triacylglycerol droplets.
Intensely colored tissues (other than the pigment cells of the skin) are rich
in and owe their color to heme and / or cytochromes. This is the case with
brown fat tissue. The color of this special type of fat tissue derives from
the abundant presence of mitochondria, which in turn contain cytochromes.
They also contain a high amount of an uncoupling protein (section 6.1) called
thermogenin.
In contrast to white fat cells, brown fat cells do not release fatty acids
into the circulation but instead perform β-oxidation themselves. The derived
hydrogen is oxidized in the respiratory chain. However, the resulting proton-
motive force is not used for ATP synthesis but instead simply dissipated as
heat – the purpose of brown fat is heat production. There is little brown fat in
adult humans or most other adult mammals. However, there is a substantial
amount in newborns, who because of their higher surface-to-volume ratio (and
their helplessness) are more prone to hypothermia. Brown fat is also found
in hibernating animal species such as polar bears, which need it to reheat
themselves to operating temperature during arousal from hibernation.
a) O O O OH
HO HO
b)
TAG
FA + glycerol Acetyl-CoA
Succinate
Acetoacetyl-CoA
TCA
FAD FADH2
Succinyl-CoA Acetoacetate
FA Acetyl-CoA
NADH+H+
NAD+ NADH+H+
NAD+
β-HB β-HB
Acetoacetate
Another condition with accelerated breakdown of fat is plain ol’ flu and
other states of fever. Metabolism is accelerated by fever; at the same time,
patients often have little or no appetite. Fat is broken down, and again an
acetone smell may develop. This by itself does not indicate a serious condition.
Utilization of ketone bodies in the brain, muscle and other tissues is straight-
forward (Figure 10.9): (1) β-Hydroxybutyrate is dehydrogenated to acetoacetate,
(2) acetoacetate steals coenzyme-A from succinyl-CoA to become acetoacetyl-
CoA, (3) thiolase cleaves acetoacetyl-CoA to acetyl-CoA.
132 10 Triacylglycerol metabolism
a) O
O CoA-SH
+
H
Enzyme S CH3
CoA S CH3
B
Enzyme S
H CH2 S CoA
B
O
O O
H3C S CoA
b) +
Enzyme B H
O O O O
CH2 S CoA
1
Enzyme-B O
H2O
H CH2 S CoA
1
CoA
O
O OH
O O
2 CoA S CH3
HO CH3 CH2 OH
Acetyl-CoA O
CH3 CH3
c)
NADH+H+ NAD+
C O HO C H
CH2 CH2
C O C O
OH OH
ATP ADP
O O OH
C
CoA S CH3 CoA S C O
CO2
has been determined using electron microscopy and image processing. The
crystal structure has recently been determined as well (Science 321:1315-22,
2008).
The growing fatty acyl chain remains covalently attached to the enzyme
throughout the entire synthesis process. Attachment is through a phospho-
pantetheine group, which provides a flexible tether, enabling the acyl chain to
travel and visit the various active sites on the enzyme in turn (Figure 10.12c).
This is reminiscent of the lipoamide moiety in pyruvate dehydrogenase and
of biotin-dependent carboxylases.7 The phosphopantetheine tether group is
actually not new to us – it also occurs in coenzyme A (Figure 10.12b).
In Figure 10.13, the structure of fatty acid synthase has been rather drasti-
cally simplified; only the two thiol groups, which serve as points of covalent
attachment of acyl-CoA and malonyl-CoA, are shown. Keep in mind, however,
that they are close to each other only in two reactions (3 and 7 in Figure 10.13).
For the other reactions, the tethered acyl residue swings back and forth to in-
teract with the other active sites. The reactions catalyzed by fatty acid synthase
are as follows (see Figure fatFAsynthesis):
1. Transfer of an acetyl-group from acetyl-CoA to the active-site cysteine.
Both in the substrate and the product, the malonyl group is bound as a thioester,
so that this reaction is easy; a similar exchange is part of the thiolase reaction
(Figure 10.10a).
2. Transfer of a malonyl group from malonyl-CoA onto the phosphopan-
tetheine group of the FA synthase. Since the two carriers are so similar, this is
again a very straightforward reaction.
3. Condensation of the malonyl group to the end of the acetyl group. Apart
from the concurrent decarboxylation (which facilitates the transient formation
of a carbanion), this reaction resembles the reversal of the thiolase reaction in
β-oxidation.
4. Reduction of the keto group to a hydroxyl group,
5. Elimination of water to create a C=C double bond and
6. Reduction of the latter to a single bond. These steps are reversals of those
occuring in β-oxidation, except that NADPH is used as the redox coenzyme.
7. Transfer of the extended acyl chain from the phosphopantheteine group
to the aforementioned active site cysteine. Note that this reaction is actually
very similar to the initial transfer of an acetyl group from acetyl-CoA – in both
cases, transfer is from a pantetheine group to the cysteine.
8. The phosphopantetheine site is now free to accept another malonyl
group, and the cycle repeats, with the acetoacetyl group now taking the place
of the acetyl group in the first cycle.
7 In E. coli, the phosphopantetheine group is associated with a separate small protein, the acyl
carrier protein (ACP). In mammalian fatty acyl synthesis, ‘ACP’ is not a separate protein but is
part of the synthase molecule itself.
10.5 Fatty acid synthesis 135
a)
*
*
b) O CH3 H
Enzyme Ser O P O C C C C N C C C N C C S
H2 H H2 H2 H H2 H2
O CH3 OH O O
O O CH3 H
Adenosine P O P O C C C C N C C C N C C S
H2 H H2 H2 H H2 H2
O O CH3 OH O O
c) O
Cys-S-
Figure 10.12 Structure of fatty acid synthase. a: Electron microscopy. Left: The
white squares highlight individual molecules. (No, I can’t recognize them either.) Right:
The three-dimensional structure, obtained by merging and averaging many images of
individual molecules. The two subunits of the dimer are elongated and oriented in
antiparallel fashion. The stars highlight the putative location of the active sites. From:
Brink, Jacob et al. PNAS 99:138-143 (2002). b: Structure of the phosphopantetheine
group (top) compared to coenzyme A. c: The phosphopantetheine group acts as a tether
for the acyl group, which can therefore visit the various active sites of the synthase.
The cysteine is located in one of the active sites (the one which has acyl transferase
activity). Only one of the two enzyme subunits is shown.
136 10 Triacylglycerol metabolism
Pant S
Enzyme Pant S
Cys S Enzyme
1
Cys S CH3
H3C S CoA
O
O
CoA S
Malonyl-CoA
2
CoA S
Malonyl-CoA
O O
7
3 CO2
Pant S CH3
Cys S Enzyme O O
Cys S
NADP+
NADPH
6
NADPH 4
NADP+
Figure 10.13 Reactions of fatty acid synthesis. “Pant” represents the pantetheine
moiety of fatty acyl synthase. See text for details.
10.5 Fatty acid synthesis 137
a) b) acetyl-CoA acetyl-CoA
ADP
Mitochondrial Cytosol
5 1 ATP
matrix
citrate citrate
acetyl-CoA acetyl-CoA
ADP oxaloacetate oxaloacetate
1
ATP malate malate 2
2
5 citrate citrate
CoA-SH
NADH+H+ NAD+ NAD+ NADH+H+
OA OA NADH+H+
ADP+Pi
2 NAD+ c) 2 acetyl-CoA 2 acetyl-CoA
4 malate
ATP NADP+ 3 CoA
3 NADPH+H+ acetoacetyl-CoA
1
CO2
2 CoA 2 ADP
CO2 pyruvate pyruvate
CoA
ATP
acetoacetate acetoacetate
Figure 10.14 Mechanisms for the transport of acetyl-CoA from the mitochondrion to
the cytosol for fatty acid synthesis. a: The textbook shuttle. Enzymes: 1, ATP-citrate
lyase; 2, malate dehydrogenase; 3, malic enzyme; 4, pyruvate carboxylase; 5, citrate
synthase. b: A simpler yet more likely shuttle. Enzymes as in a). c: The acetoacetate
shuttle. 1: reactions of ketogenesis; 2: acetoacetyl-CoA synthetase; 3: thiolase.
Finally, when the full length of 16 carbons has been reached, the palmitate
residue is hydrolyzed off the pantetheine residue instead of being transferred
to the cysteine (not shown).
The dimeric nature of the protein raises two possibilities for its mechanism:
(1) The two subunits operate independently, that is the acyl chain only visits
active sites within the subunit to which it is bound. (2) The acyl chain undergoes
reaction in one or more of the active sites on the other subunit.
Splitting the dimer into monomers leaves most of the enzyme activities
intact, indicating that these reactions occur within the same subunit. However,
the initial reaction in each cycle—the condensation reaction between the fully
reduced acyl chain and the next malonyl residue—only occurs in the dimer,
which suggests that it may be catalyzed by the other subunit.
the problem of getting it out of the mitochondrion and into the cytosol.
You may recall that there was a similar problem in β-oxidation. In that case,
the transport of acyl-CoA was accomplished by the carnitine carrier system
(section 10.2.1). Since all reactions in the latter system are reversible and acetyl-
CoA is also an acyl-CoA, we might assume that the carnitine carrier system
could also help us out here. However, if it did transport both acetyl-CoA and
longer acyl-CoAs in both directions, it might set up a futile cycle of fatty acid
synthesis and degradation. Accordingly, acetyl-CoA is transported by other
means. There seem to be several pathways that contribute to this transport
(Figure 10.14):
1. The major mechanism uses the cytoplasmic enzyme ATP-citrate lyase.
Citrate is exported by the tricarboxylate carrier system and cleaved; concomi-
tantly, the acetate fragment is activated to acetyl-CoA using ATP. The problem
then arises how to dispose of the oxaloacetate. While the textbooks usually
account for this as shown in Figure 10.14a, this mechanism is not only com-
plicated but also unbalanced. Citrate can only be transported in exchange
for something. One possibility is malate, which suggests that the mechanism
outlined in Figure 10.14b is more reasonable.
A fairly straightforward transport mechanism that is active at least in mice, but
probably also other mammals, is the export of acetoacetate, which is generated
in the mitochondria by the ketogenesis pathway (Figure 10.10). Acetoacetate
can be converted back to acetyl-CoA by cytosolic acetoacetyl-CoA synthetase,
which uses ATP, and a thiolase (Figure 10.14c).
The acetoacetate shuttle has the advantage of the lowest ATP expenditure
(one ATP per two molecules of acetyl-CoA translocated).
citrate
NADPH+H+
NADPH+H+ α-ketoglutarate α-ketoglutarate + CO2
succinyl-CoA
consumed in a futile cycle as discussed then, this isocitrate can again be made
available for cytosolic NADPH regeneration.
continued excess calory intake – what is going to become of the surplus acetyl-CoA if fatty acid
synthesis is inhibited: Ketone bodies? Cholesterol? Will glycolysis be backed up and diabetes be
induced? All of the above?
140 10 Triacylglycerol metabolism
a)
OH
O O
+
H
O S Cys Enzyme
NH2
O O
OH S Cys Enzyme
NH2
O O
b)
avg. tumor volume (mm3)
OH
500
HO OH
400 untreated
300
O O
200
O 100 compound 7
OH
O 0
OH 0 10 20 30 40
compound 7 OH days of treatment
Cholesterol metabolism
141
142 11 Cholesterol metabolism
Table 11.1 Characteristics of the major lipoproteins. VLDL: Very low density lipopro-
tein; LDL and HDL: Low and high density lipoproteins.
CH2OH
H3C CH3
C O
CH3 HO CH3
CH3 OH
CH3 CH3
H H H H
HO O
Cholesterol Cortisol
O CH3
C OH
CH3 CH3
CH3
H H H H
O HO
Progesterone Estradiol
O
O
OH
OH
CH3
CH3
CH3
CH3
H H
O
HO O
deficiency of this receptor is associated with increased blood levels of LDL and
increased risk of heart attack and stroke (see section 11.9).
Excess cholesterol can be transported back from peripheral tissue to the liver
by high density lipoprotein (HDL). Among all lipoproteins, this one has the
highest ratio of protein to lipid and therefore the highest density. A high
level of HDL in the blood is associated with a decreased risk of cardiovascular
disease; therefore, HDL has been dubbed the ‘good cholesterol’ as opposed to
LDL, the ‘bad cholesterol’. The liver can either recycle the cholesterol into new
144 11 Cholesterol metabolism
polymerization.
11.5 Synthesis of cholesterol 145
a) CH3
+ H3C CH3
H3C N CH3
CH3
CH3
O H CH3
H H
O O O
O O O
LCAT
b) free cholesterol
O O
O
cholesterol ester
O
that from this point onwards the substrates have no other option than becom-
ing a sterol.4 Therefore, HMG-CoA reductase is the main target of regulatory
4 No quantitatively important one, at least. However, farnesyl-pyrophosphate is also used in
146 11 Cholesterol metabolism
HMG-CoA-
reductase
Acetyl-CoA HMG-CoA Mevalonate (C6)
3 ATP
3 ADP CO2
Figure 11.3 Overview of cholesterol biosynthesis. The first committed step is the
reduction of HMG-CoA to mevalonate, which then is activated by ATP for polymeriza-
tion up to squalene. Squalene, the last linear metabolite, is converted to lanosterol, the
Mother of All Sterols.
3
CO2 P
5
P-P
NADPH+H+
7
P-P P-P NADP+
NADPH+H+ O2 squalene
8
NADP+ H2O
+
C
HO HO lanosterol
O
+
H
9
Figure 11.4 Enzyme reactions in the synthesis of lanosterol, the sterol precursor
of cholesterol. PP stands for pyrophosphate. Enzymes/reactions: 1, HMG-CoA reduc-
tase; 2: Mevalonate kinase and phosphomevalonate kinase; 3: Phosphomevalonate-5-
pyrophosphate decarboxylase; 4: Isopentenyl-pyrophosphate isomerase; 5: Geranyl-
pyrophosphate synthase; 6: Farnesyl-pyrophosphate synthase; 7: Squalene synthase; 8:
Squalene monooxygenase. 9: Reaction intermediates of the squalene monooxygenase
reaction.
148 11 Cholesterol metabolism
Mitochondria
all proteins, are at least partially polar and cannot immerse completely inside
the same apolar environment. The solution to this problem is to perform the
reactions at the interface of polar and apolar environments, that is at membrane
surfaces. This means that the membrane of the smooth ER is not just there to
delimit a separate compartment, but itself is a reaction compartment.
The membrane of the smooth endoplasmic reticulum is extensively bulged
and invaginated, which gives it a very large surface area. The throughput of
cholesterol synthesis should be proportional to this surface area. Accordingly,
the development of the smooth ER is particularly prominent in cells that per-
form sterol or steroid hormone synthesis (Figure 11.5).
b)
ER
Golgi
c)
ER
Golgi
NM
SRE
+
RNA polymerase
HO HO
COOH COOH
OH OH
O
4. In the nucleus, SREBP binds to sterol response elements (SREs). These are
short specific DNA sequences that occur in various places of the genome.
5. One SRE is located upstream of the gene of HMG-CoA reductase. Binding
of SREBP to this element increases transcription and therefore the overall
activity of HMG-CoA reductase.
Induction of HMG-CoA reductase will increase the rate of cholesteorl synthe-
sis, which will ultimately suppress the further proteolytic activation of SREBP.
The main factors that may induce a high plasma cholesterol concentration
are (1) the diet: Rich in cholesterol, or in fat or excess carbohydrates, which may
be converted first to acetyl-CoA and then to cholesterol, (2) physical inactivity:
Sedentary lifestyles, such as typical of prison or university inmates, (3) genetic
factors, in particular a defect of the LDL receptor.
The latter condition is particularly serious if it is homozygous, that is if the
receptor is completely absent. The levels of LDL and cholesterol in the plasma
can reach several times the normal level. The rate of progress of atherosclerosis
and the risk of stroke and myocardial infarction are greatly increased, so much
so that the typical life expectancy of homozygous patients is only 30-40 years.
Heterozygous patients still are at significantly higher risk than the average
person, but the condition is more tractable with the available means of therapy.
exposes hydrophobic and cationic binding sites, which combine avidly with the
hydrophobic and anionic moieties of bile acid molecules. Ingested cholestyra-
mine will bind bile acids and simply take them along down the pipe. To replace
the lost bile acids, the liver will increase their synthesis and therefore deplete
the pool of cholesterol.
Apart from bile acids, excess cholesterol itself is also secreted into the bile,
where it depends on bile acids to remain in solution. A drawback of cholestyra-
mine therapy is that, by depleting bile acids, it promotes the precipitation of
cholesterol, favouring the formation of cholesterol bile stones.
While for maximum effect the different principles are often combined, inhi-
bition of synthesis with statin drugs is the most effective single principle and
currently has a prominent place in therapy.
Chapter 12
We have seen before that during digestion in the intestines proteins are bro-
ken down to their constituent amino acids. Proteins contain twenty standard
amino acids, which are incorporated into them during translation, and several
non-standard ones that are mostly formed by post-translational modification.
These are much less abundant than the standard amino acids and will not be
considered here.
Amino acids, in human metabolism, have three main usages:
1. As building blocks for our own protein synthesis. Animals, including
humans, are essentially parasites and have a lazy synthetic metabolism. Ac-
cordingly, we possess synthetic pathways for only 10 out of the 20 standard
amino acids. The residual 10 amino acids have to be obtained from the diet
and are called the essential amino acids.
2. As a source of energy. Depending on the composition of the diet, this
role may be very significant. Carbohydrates occur nearly exclusively in plant-
derived foodstuffs. Therefore, on a meat-only diet, amino acids become the
major source of glucose.
3. As building blocks for other things such as nucleotides and heme.
We will here focus energy metabolism, that is on the degradation of amino
acids. Synthesis will be skipped, except for some simple examples that involve
no more than transamination. We only note that nine out of twenty amino acids
cannot be synthesized in mammalian cells and therefore have to be obtained
with the diet. These essential amino acids are histidine, isoleucine, leucine,
lysine, methionine, phenylalanine, threonine, tryptophane, and valine. Arginine
is synthesized but apparently not in sufficient amounts and often listed as a
tenth essential amino acid.
154
12.1 Overview of amino acid degradation 155
Figure 12.1 Overview of amino acid degradation. Glucogenic amino acids that give
rise to either pyruvate or TCA intermediates that can be turned into glucose. Ketogenic
amino acids give rise to acetoacetate and acetyl-CoA, which do not yield glucose.
All amino acidsdegradation amino acids contain at least one nitrogen atom,
which forms their α-amino group.Nitrogen has no use in energy metabolism
and has to be eliminated. There are two key processes in metabolic nitrogen
elimination: 1. Transamination removes the α-amino group from one amino
acid and transfers it to α-ketoglutarate. This leads to the accumulation of
glutamate. 2. The release of nitrogen from glutamate and its conversion to
urea. This is accomplished by the urea cycle in the liver.
Several amino acids—arginine, asparagine, glutamine, histidine, lysine, pro-
line, tryptophan—contain additional nitrogen atoms in their side chains. For
these, adapter pathways exist that ultimately also feed the nitrogen into the
urea cycle.
Removal of nitrogen is typically an early step in amino acid degradation and
leaves behind the carbon skeleton. The structure of the latter is different for
each amino acid, and accordingly each amino acid has its specific pathway of
degradation. However, they can be grouped into two broad classes. As Figure
12.1 shows, most amino acids can be converted to intermediates of the citric
acid cycle or to pyruvate. These are called glucogenic amino acids, since they
can contribute to the synthesis of glucose (gluconeogenesis). Those that yield
acetoacetate are called ketogenic, since acetoacetate is one of the two ketone
bodies (see section 10.4).
What happens to glucogenic amino acids when they are available in excess
over the demand for glucose? This may well happen if we are on a very protein-
rich diet. One option would be to first convert all substrates to oxaloacetate
156 12 Amino acid metabolism
in the citric acid cycle and then short-circuit gluconeogenesis and glycolysis at
the level of phosphoenolpyruvate:
12.2 Transamination
In the degradation of most standard amino acids, an early step in degrada-
tion consists in transamination, which is the transfer of the α- amino group
from the amino acid to an α-keto acid. There are several different aminotrans-
ferases, each of which is specific for an individual amino acid or for a group
of chemically similar ones, such as for example the group of branched amino
acids, which includes leucine, isoleucine, and valine. With all these enzymes,
the α-keto acid that accepts the amino group is always α-ketoglutarate (Fig-
ure 12.2). Transamination is freely reversible; therefore, both glutamate and
α-ketoglutarate are substrates of every single transaminase. If amino groups
are to be transferred between two amino acids other than glutamate, this will
still occur by transient formation of glutamate (Figure 12.2b).
The mechanism of transamination is depicted in Figure 12.4 for alanine, yet
is the same with all transaminases. The reaction occurs in two stages:
1. Transfer of the amino group from alanine to the enzyme, which releases
pyruvate, and
2. Transfer of the amino group from the enzyme to α-ketoglutarate, which
releases glutamate.
In Figure 12.4, only the first half-reaction is shown. The second half-reaction is
the exact reversal of the first one; this also implies that the entire reaction is
reversible. Overall, the mechanism consists in the first substrate arriving and
leaving before the second substrate enters and leaves; this is dubbed a ping
12.2 Transamination 157
C O H2N CH H2N CH C O
CH2
+ CH3 CH2
+ CH3
CH2 CH2
COOH COOH
H2N CH C O H2N CH
CH2 COOH
COOH
H 2N CH COOH
COOH CH2 C O
C O CH2 CH2
CH3 COOH COOH
pong bi bi reaction (Figure 12.3). 1 While two different substrates must be used
for the reaction to have a net effect, it is of course possible for amino acid 1
and amino acid 2 to be identical – the reaction will work just fine, but simply
achieve no net turnover.
The reaction mechanism revolves around the coenzyme pyridoxal phos-
phate (PLP):
1. At the outset of the reaction, PLP is bound as a Schiff base to the -amino
group of a lysine residue in the active site (Figure 12.4a).
2. The bond between PLP and the enzyme is separated, and PLP forms a
Schiff base with the amino acid substrate instead (Figure 12.4b, steps 1 and 2).
3. The liberated lysine residue abstracts the α hydrogen as a proton (step
3), and the electron left behind travels all the way down to the nitrogen at the
bottom of the PLP ring. PLP is often said to act as an electron sink. This has
the effect of turning the bond between the α carbon and the α nitrogen into a
Schiff base.
1 The mind boggles when one tries to figure out what kind of greek nomenclature Old World
biochemists could have dreamed up for this behaviour. Maybe something like: Amphiballistic
antidromic sequential?
158 12 Amino acid metabolism
Figure 12.3 The ping pong bi bi kinetic mechanism of transamination. The two
substrates arrive and leave one after another; the second half-reaction (dotted blue
arrow) is the exact reversal of the first one (solid blue arrow), save that a different
α-keto is used.
4. The Schiff base is hydrolyzed to yield the α-keto acid and the amino
derivative of PLP, called pyridoxamine phosphate (steps 4 and 5).
The PLP in its various forms stays within the active site throughout, even
while not bound to the enzyme covalently. As stated above, the second half
reaction is the exact reversal of the first, and a good exercise for you would be
to draw the individual steps yourself.
ammonia derived from bacterial metabolism in the large intestine. Antibiotics are used in this
condition to reduce bacterial growth and ammonia formation.
12.3 The urea cycle 159
HC + 1 HC +
N CH3 N CH3
H H
Enzyme
2
Enzyme
CH2
CH2
H O +
NH3 H NH2
H3C C COOH 3 H3C C COOH
+
H N N
CH CH
H2 H2
C O C O
P P
4
HC HC +
N CH3 N CH3
H H
5
H O O
HC HC
N CH3 N CH3
H H
160 12 Amino acid metabolism
O P O
OH O
O ATP ADP O O ATP ADP
O O
C O C NH2
C O P O
H2N C O
HO O P
HO OH
1 NH3
1 OH 1 2
OH
NH2
CH2 A P P P CH2
NH CH2 NH NH2
HC N C CH2
HC NH2 C O AMP C O
H
COOH HC NH2
NH COOH NH NH
COOH
CH2 CH2 CH2
CH2
3 CH2 P P
3 CH2
3
CH2 CH2 CH2
4
COOH NH2 NH2
CH H2N C H2N C
H2O
CH N O NH2
CH2 5 CH2
CH2 CH2
HC NH2 HC NH2
COOH COOH
α-Keto acid 1
Amino acids 1 and 2
-
Glutamate
HCO3
NH3 α-Keto acid 2
2 α-Ketoglutarate
Carbamoyl-P
Aspartate Glutamate
Citrulline
Oxaloacetate
Fumarate
Arginine
Urea
Figure 12.6 Cooperation of citric acid cycle, urea cycle, and transamination reactions
in urea synthesis. Typeset in bold are the names of the only metabolites for which
there is a net turnover. All other metabolites are part of closed cycles.
a)
O OH O OH O OH O OH
O OH O OH O OH O NH2
b) c)
O OH O OH
COOH COOH
NAD+ NADH+H+
H2N CH C O H2N CH H2N CH
d) O OH O OH
ATP ADP
H2N CH H2N CH
CH2 CH2
O OH O NH2
Figure 12.7 Auxiliary reactions in nitrogen transport from peripheral tissues to the
liver. a: Glutamate synthase (GOGAT) disproportionates glutamate to α- ketoglutarate
and glutamine. This happens in the periphery. b, c: Glutamate dehydrogenase (b) and
glutaminase (c) release ammonia from glutamate and glutamine, respectively. This
happens mostly in the liver. d: Glutamine synthetase scavenges excess ammonia. This
occurs both in the liver and in peripheral tissues.
the reversal of this reaction—glutamate synthase. Note that this enzyme pro-
duces an amide group, not an amine. The second product of this reaction is
α-ketoglutarate, which can participate in another round of transamination. Glu-
tamine travels to the liver, where its δ-amido group is released as ammonia by
glutaminase. The glutamate produced returns to the peripheral tissue (Figure
12.8a).3
Alanine is formed from pyruvate by transamination in the peripheral tissues.
It travels through the blood to the liver, where the nitrogen is abstracted by
transamination back to pyruvate. Gluconeogenesis then turns pyruvate into
glucose, which is returned to the periphery. This process is called the glucose
3 One might think that the glutamate could be further deaminated by glutamate dehydrogenase,
a)
Glutamine Glutamine
H 2O
3
NH3
2
Glutamate Urea
Amino acid α-Ketoglutarate
b)
Alanine Alanine
α-Keto- α-Ketoglutarate
Amino acid
glutarate 4 5 H 2O
4 Glutamate
1
NH3
Pyruvate
Urea
α-Keto acid Glutamate Pyruvate
Glucose Glucose
NH3 is high -
urea cycle runs at speed
glutamine synthetase:
NH3↓
To systemic circulation
Figure 12.9 Distribution of enzyme activities related that release or capture ammonia
within the liver lobule. Ammonia is released from glutamine and glutamate in the
periphery of the lobule, which increases throughput of the urea cycle. Remaining
ammonia is scooped up and stored away as glutamine before the blood re-enters the
general circulation.
nitrogen transported, two ATP molecules are expended, since each round of
gluconeogenesis and glycolysis transports two nitrogens and consumes 4 mo-
lecules of ATP.
The toxicity of ammonia requires its concentration to be kept very low in the
systemic circulation. On the other hand, for the urea cycle to run at speed,
the concentration must be high enough to saturate the initial enzyme, car-
bamoylphosphate synthetase, to a useful degree. Therefore, ammonia must
be released as the blood enters the liver tissue, and scooped up again before
the blood is drained away into the general circulation. To make this work, the
enzymes that release ammonia or fix it are strategically distributed in the liver
tissue (Figure 12.9). We had seen before that the liver has a honeycomb-like
fine structure, with the blood filtering through the individual hexagonal lobuli
from the periphery towards the center (Figure 1.10). Glutaminase and gluta-
mate dehydrogenase, which release ammonia, are found predominantly in the
periphery of the lobule. This results in an increased concentration of ammonia
in the bulk of the lobule tissue, where the urea cycle can therefore run at speed.
Scavenging the remaining ammonia is the job of glutamine synthetase, which is
mainly found around the central veins of the lobule. This illustrates nicely how
12.5 Degradative pathways of individual amino acids 165
O OH O OH
H2N CH H2N CH
CH2 CH2
H2O NH3
O NH2 O OH
For some amino acids the degradation is a very straightforward issue. For
example, with alanine, aspartate and glutamate, transamination is all that is
required to turn them into mainstream metabolites. Glutamine and asparagine
can be accommodated by one preceding deamidation, catalyzed by glutaminase
(Figure 12.7b) and asparaginase, respectively.
Asparagine is not an essential amino acid, meaning that it can be synthe-
sized by human cells; the enzyme responsible for this, asparagine synthetase,
uses glutamine as the amide group donor. However, in some forms of leukemia,
the leukemic cells are apparently short of synthetic capacity for asparagine.
This can be exploited for therapy: Patients are treated with intravenous ap-
plication of asparaginase. 4 This lowers the serum level of asparagine and
therefore starves the leukemic cells. However, this is only one component in
the therapy – cytostatic drugs have to be added to make the therapy effective.
For the other amino acids, degradation is more elaborate. We will now consider
a few examples.
4 The enzyme is purified from the bacterium Escherichia coli. In healthy patients, injection
of a bacterial protein would rapidly induce antibodies, which would quickly render the enzyme
inactive. However, in leukemia patients, the disease itself and the cytostatic drugs simultaneously
applied conspire to suppress antibody formation. If it occurs anyway, enzyme prepared from
another bacterium, Erwinia chrysanthemi, can be used. Immunogenicity of the enzyme can be
reduced by derivatization of the protein with polyethylenglycol (PEG).
166 12 Amino acid metabolism
hydroxymethyltransferase removes
the side chain carbon as formalde- b)
hyde and immediately captures it H
onto tetrahydrofolic acid (THF). The H O CH2 C COOH O CH2 + CH2 COOH
remainder is glycine. c: Transamina- NH3
+
NH2
tion to hydroxypyruvate, reduction THF
aKG Glu
H2N CH O
H2C OH H2C OH
NADH+H+
NAD+
O OH O OH
HC OH HC OH
H2C O P ADP ATP H2C OH
Both serine and glycine are non-essential amino acids, that is they can be both
synthesized and degraded in human metabolism. The formation of glycine
actually occurs in one of the degradation pathways for serine. These two amino
acids are interesting because their degradation is the prime source of single-
carbon groups in metabolism. These ‘C1 -units’ are bound to the coenzyme
tetrahydrofolic acid, which then donates them in a variety of reactions, for
example in the biosynthesis of nucleotides. For serine, there are several routes
of utilization (Figure 12.11):
1. The most straightforward one consists in its conversion to pyruvate by
serine / threonine dehydratase (Figure 12.11a). Note that in this case the nitro-
gen is released as ammonia but not by transamination. The pathway therefore
preferably occurs in the liver, which can dispose of the ammonia. The enzyme
uses the same coenzyme (pyridoxalphosphate) and a similar mechanism as the
transaminases (Figure 12.13).
2. Serine hydroxymethyltransferase cleaves serine into glycine and formalde-
hyde. Formaldehyde, which is highly reactive, is not released by the enzyme
but is immediately fixed onto the cosubstrate tetrahydrofolic acid.
3. An alternative pathway transaminates serine to hydroxypyruvate, which
is then reduced to glycerate and phosphorylated to 3-phosphoglycerate. While
both pyruvate and 3-phosphoglycerate can serve as substrates for gluconeoge-
12.5 Degradative pathways of individual amino acids 167
a)
Enzyme Enzyme Enzyme
E-PLP
H2N CH2 COOH
b) H
H 2N N N
N
N O COOH O
H
OH N C N C C C C OH
H H H H2 H2
n
H H
N N
N N
H N N
CH2 CH2
O H HO H
H
H2N N N
N
N O COOH O
OH H2C N C N C C C C OH
H H H2 H2
H2O n
Enzyme
Enzyme Enzyme
CH2
CH2 CH2
NH2
+
NH2 NH2 H2O
H H
HO CH2 C COOH H2C C COOH
HO CH2 C COOH
+ +
HN + HN
HN
CH CH
CH H2
H2
H2
C O C O
C O
P P
P
HC HC
+ HC +
N CH3 N CH3
H N CH3 H
H
H2O
+
H E-PLP
NH3 H2O
O NH NH2
Figure 12.13 Catalytic mechanism of serine dehydratase. While the overall reaction
consists in the elimination of ammonia, the enzyme itself actually eliminates water,
which is reflected in its name. The aminoacrylate released by the enzyme is then
spontaneously hydrolyzed to pyruvate and ammonia.
and primes it for the reaction; the specific bond within the substrate that is
broken is then determined by the point of attack of some specific side chains
within each enzyme’s active site.
We have already seen that glycine is a product of serine hydroxymethyl-
transferase. Since that enzyme reaction is reversible, glycine can actually be
converted to pyruvate or 3-phosphoglycerate via serine and thus serve as a
substrate in gluconeogenesis. The required additional carbon, in the form of
N,N’-methylene-THF, can be provided by the glycine cleavage system, an enzyme
that completely breaks down glycine (Figure 12.14). Therefore, two glycines
are required to provide one pyruvate. The glycine cleavage system again uses
pyridoxalphosphate in its catalytic mechanism, and as far as I can see the PLP
again functions in a similar way as described before.
NAD+ NH3
NADH + H+
methylene-
NH3+CO2 glycine
THF
Figure 12.14 Glycine catabolism. The glycine cleavage system completely breaks
down glycine, extracting one methylene group as N,N’-methylene-tetrahydrofolate. This
methylene group can be used by serine hydroxymethyltransferase to convert a second
glycine molecule to serine and then to pyruvate.
C C CH2 CH
H3C H CH3
1 H3C H CoA 2 CO2 3
CH3
C
H3C H H3C CH3
CH3
CO2
CoA CoA CoA 4
H2O ATP
S S S
C O C O C O
ADP+Pi
CH3 6 CH2
5 CH
Figure 12.15 Degradation of leucine. The reactions are numbered in accordance with
the descriptions in the text.
2
COOH
Glu
C O
OH COOH CH2
CH2 CO2 H 2O O2
HC HC CH
HC CH
3 HC CH
OH O2 Ascorbate-H2 OH
4 Ascorbate
OH
COOH
O O O
O
HC CH2 HO H
O C C C C
C C C OH
HC H H2 H2
CH2
C 5 O
O
6 H 2O
O O O
H
HO C C H C
C C OH C C OH
H H2 H2
O
12.6.1 Phenylketonuria
COOH
Phenylpyruvate C O
CH2
HC CH
HC CH
Tyrosine- C
H
aminotransferase
Figure 12.18 Principle of the Guthrie test for phenylketonuria. See text for additional
details.
2. A small piece of filter paper, soaked with a drop of test blood, is placed
on the surface of the agar.
3. Small molecules including amino acids will diffuse from the filter paper
into the surrounding agar. If the test sample contains a high level of pheny-
lalanine, as is the case with infants that suffer from phenylketonuria, bacterial
cells right next to the filter paper will gobble it up and start growing.
4. If very little phenylalanine is available, as will be the case with healthy
children, no bacterial growth will occur around the samples.
Therefore, a zone of bacterial growth around the filter paper snippet will
identify a sample from a patient with phenylketonuria. Note that, for this test
to work, we cannot collect the blood sample right away after birth. In the
womb, the fetal blood equilibrates with the mother’s, and so the phenylalanine
174 12 Amino acid metabolism
a) Tyrosine ↑
p-Hydroxyphenylpyruvate ↑
Homogentisate ↑
fumarylacetoacetase
Fumarate + acetoacetate
b) Tyrosine ↑
p-Hydroxyphenylpyruvate ↑
p-Hydroxyphenylpyruvate dioxygenase
Homogentisate O O NO2
Maleylacetoacetate
O CF3
Fumarylacetoacetate
(Nitro-trifluoromethyl-benzoyl)-
cyclohexanone (NTBC)
Fumarate + acetoacetate
Figure 12.19 Tyrosinemia type I. a: Enzyme defect and resulting metabolic backup
in in tyrosinemia type; b: Therapy with the enzyme inhibitor nitro-trifluoromethyl-
benzoyl-cyclohexanone (NTBC).
Chapter 13
13.1 Insulin
The insulin most widely known hormone in metabolic regulation is certainly
insulin, which facilitates the utilization of glucose. This hormone is so named
because it is secreted in the pancreatic islets (insula = latin for island, islet).
These islets are small portions of tissue that are found interspersed within the
176
13.1 Insulin 177
c)
d)
sea of the exocrine pancreas. The exocrine1 pancreas, which accounts for 98%
of the entire mass of the organ, is responsible for secreting all the enzymes and
bicarbonate we have encountered before. The islets, in contrast, are endocrine
and secrete a variety of peptide hormones into the blood stream, most notably
insulin and glucagon.
The importance of the pancreas in the regulation of blood glucose was rec-
1 Exocrine means that the secretion is targeted to the ’outside’ world (and that would include
the intestinal lumen), whereas endocrine secretion is targeted into the blood stream. Thus, all
hormones are secreted by endocrine glands, and the branch of medicine that specializes in
hormones, their glands and related diseases is endocrinology.
178 13 Hormonal regulation of metabolism
2 Charles Best did not officially receive the Nobel Prize; instead, Banting shared it with John
MacLeod, another researcher at U of T. However, Frederick Banting declared that Best should
have been included and split his share of the prize with him to affirm this point. Thanks to
Alastair Brownie for correcting this historical detail.
13.1 Insulin 179
Cambridge managed to develop the first workable methodology for protein se-
quencing and to elucidate the primary structure of insulin, which thus became
the first protein with completely known amino acid sequence. The primary
structure of insulin is depicted in Figure 13.2. It consists of an A chain and a
B chain, which are derived from a common precursor that undergoes a series
of site-specific cleavages. The final products, insulin and the C peptide, are
both stored in secretory vesicles and are released together. Note the sequence
variations between the bovine, the porcine and the human insulin. Because
of these sequence variations, it is possible for diabetics to develop antibodies
against the animal insulins; this is a major reason why nowadays recombinantly
produced human insulin is preferred for the treatment of diabetes.
+
sent transmembrane helices. Insulin
ADP+Pi
The ATP binding folds (ABF) Membrane
are on the cytosolic side of potential
K+
the membrane. c: Binding
of ATP will cause a confor- ATP
H2O + CO2
mational change to the sul-
fonylurea receptor, which in
turn will cause the channel to
close. This will partially de-
polarize the membrane and b) Kir-Channel Sulfonylurea receptor
induce insulin secretion via
opening of a voltage-gated
calcium channel. d: Structure
of tolbutamide, an oral drug
that binds to the sulfonylurea
receptor and is used in type
II diabetes. The sulfonylurea ABF ABF
moiety is highlighted.
c)
K+
ATP ATP
ATP
O O
d)
H3C S N N C C C CH3
H H H2 H2 H2
O
phosphorylation of insulin receptors locks them in the active state, from which
they will only revert to the inactive state upon dephosphorylation by a protein
tyrosine phosphatase, even if insulin leaves the receptor again.3
Apart from this mutual phosphorylation, the insulin receptor kinase acts
3 However, it seems that insulin stays bound to the receptor for very long periods of time.
13.1 Insulin 181
a) Ligand
Receptor domain
(extracellular)
Enzyme domain
(intracellular)
b)
ATP ADP
P P
P P
IRS-1 IRS-1 P
ATP ADP P
P
IRS-2(3,4) IRS-2(3,4) P
ATP ADP P
Figure 13.4 The insulin receptor. a: The receptor is a transmembrane protein that
has an extracellular ligand-binding domain and an intracellular protein kinase domain;
the kinase is activated by binding of insulin. b: Ligand-activated insulin receptors will
first perform mutual phosphorylation and then phosphorylate a series of intracellular
regulatory proteins, the so-called insulin receptor substrates (IRS).
P-Glycogen synthase
kinase 3 (inactive)
b) I I d)
I I
I I
I I
Transport of GluT4 to cell
surface by vesicle fusion
PP Phosphorylation of nuclear factors,
induction of genes for glycolytic PP
enzymes etc.
IRS-1 IRS-1 P Grb-2
PI-3K P IRS-1 IRS-1
Sos MAPK-1-P
GTP
displaces Phosphorylation of
Ras MEK-P PIP2 PIP3 PKB
GDP cytoskeletal proteins
Raf-1 MAPK-1
MEK
Figure 13.5 Intracellular effects of insulin receptor stimulation. a: One of the pathways that lead to activation of glycogen
synthesis. b: Mediation of transcriptional effects of insulin. c: Translocation of glucose transporters from intracellular vesicles
to the cytoplasmic membrane. d: Movement of glucose transporters is mediated by protein phosphorylation, too. (See text for
further details.)
183
184 13 Hormonal regulation of metabolism
Ligand
extracellular space N
‘7TM’ - receptor
cytosol
γ β α GTP
γ β α heterotrimeric
G-protein
GDP
GDP
N N
inactive active
γ β α Pi γ β α
GDP GTP
I personally prefer the idea that this complexity serves the purpose of allow-
ing different signaling pathways to interact and still yield one coherent, sensible
response. The network of intracellular kinases, phosphatases and adapter pro-
teins constitutes the brain of the cell, and a brain just needs a certain level of
complexity.
Adenylate cyclase
α
ATP
GTP γ β
cAMP cAMP
R R cAMP R R
cAMP cAMP
C C
Protein kinase A,
Protein kinase A, inactive C C active
3. Upon binding of GTP, the α-subunit dissociates from the βγ-dimer and
binds to its effector protein.
4. After a certain amount of time, the GTPase activity that is built into the
α-subunit cleaves GTP to GDP.
5. Cleavage of GTP causes the α-subunit to revert to its inactive conforma-
tion, to leave its target protein, and to re-associate with the βγ-dimers.
The system thus returns to its resting state.
Both the epinephrine receptor More specifically, the β-adrenergic receptor,
of which there again are several subtypes. and the glucagon receptor are cou-
pled to the same G protein, which binds to adenylate cyclase as its effector
protein and activates itadenylate cyclase. Because of this, the metabolic effects
on a cell that has receptors for both of them will always be similar. Differences
between the effects of the two hormones arise from the fact that the receptors
have different tissue distributions; many cells have receptors for epinephrine
but not glucagon.
The consequences of the stimulation of adenylate cyclase are depicted in
Figure 13.7. Protein kinase A, as we have seen before, has several effects:
1. It phosphorylates and thereby inactives glycogen synthase.
2. It also phosphorylates phosphorylase kinase, which in turn phosphory-
lates and thereby activates glycogen phosphorylase, so that more glucose gets
released from glycogen.
186 13 Hormonal regulation of metabolism
COOH
Figure 13.8 Nuclear a)
H2 N CH
hormone receptors and
CH2
transcriptional regulation. CH2OH
C O
a: Structures of cortisol (left) CH3
HO OH
and of the thyroid hormone I I
triiodothyronine (right). The CH3
O
iodines contained in this H H
mRNA
Figure 13.9 Obese mice, which are homozygously deficient for leptin, can be brought
to normal weight by way of leptin substitution. (Image ripped off from http://www.
biochemsoctrans.org/bst/033/1063/bst0331063f01.gif.
example, cortisol and cortisone induce enzymes for glycogen metabolism and
gluconeogenesis but also receptors for epinephrine. Major effects of cortisol
action are:
1. Stimulation of protein breakdown. The amino acids released are largely
used for gluconeogenesis.
2. Increased enzyme activities for glycogen synthesis and breakdown (!), and
for gluconeogenesis.
3. Increased sensitivity to epinephrine and norepinephrine.
4. Suppression of pain and inflammation by inhibition of prostaglandin syn-
thesis.
Cortisol is often considered a chronical stress hormone, in contrast to the
acute stress hormones epinephrine and norepinephrine.
Thyroid hormones also have a variety of regulatory targets. One key effect is
the induction of thermogenin, which is simply a respiratory chain uncoupling
protein. The mode of action of uncoupling proteins has been described in
section 6.1. The up-regulation of uncoupling proteins is responsible for the
finding of accelerated metabolism, hyperthermia and weight loss in patients
with excessive thyroid hormone secretion (hyperthyreosis).
13.4 Leptin
A relatively recently discovered hormone is leptin. This peptide hormone is
released by fat cells and taken up by the hypothalamus, where it restricts
the appetite. The hypothalamus has a key role in the homeostasis of many
physiological parameters and controls the autonomic nervous system as well
as the hypophyseal gland, which in turn controls the activity of the thyroid and
adrenal glands. The major variable that controls leptin secretion is simply the
volume of fat tissue, which means that leptin regulates metabolism on a much
longer time scale than the other hormones discussed above.
The discovery of leptin was accomplished with two different mice strains
that lack the hormone, named “obese mice”, or the receptor, named “diabetic
188 13 Hormonal regulation of metabolism
mice”. Despite these names, both strains show the same combination of over-
weight and type II diabetes. While the receptor-deficient strain unsurprisingly is
resistant to leptin, the hormone-deficient one reacts to the hormone in a quite
impressive fashion (Figure 13.9. Leptin deficiency is not a common condition
in humans, however, so that the therapeutic potential of leptin substitution is
limited.
Chapter 14
Diabetes mellitus
To conclude this course, we will try and apply what we have learned to un-
derstand the metabolic phenomena underlying the most common metabolic
disease, diabetes mellitus.
The name ‘diabetes mellitus’ means ‘honey-sweet flow-through’.1 That tells
us about two things:
1. An important symptom of the disease: Copious flow of urine, containing
significant amounts of glucose.
2. A classical method of diagnosis. The alternative to diabetes mellitus is
diabetes insipidus (insipid, flavourless), which is caused by a deficiency of the
antidiuretic hormone due to lesions in the posterior lobe of the hypophyseal
gland and is not related to glucose metabolism.
If you plan on going on to medical school, you will be glad to learn that the
classical method of diagnosis is no longer used in clinical practice.
1 For geeks: More precisely, ‘marching through’ – you may recall the term ‘adiabatic’ from
thermodynamics, meaning ‘without permeation’, which is the opposite of ‘diabetic’. The syllable
also occurs in the title of Xenophon’s work Anabasis, or Invasion, which details his experiences
as a Greek mercenary in the ancient Persian empire. This work served Alexander the Great in the
preparation for his campaign into Persia.
189
190 14 Diabetes mellitus
GR β-AR IR
AC
Phosphodiesterase 3
Fructose-2,6-bis-P
Pyruvate dehydrogenase
PFK-1 Fructose-1,6-bisp’ase
GR β-AR IR
AC
Phosphodiesterase 3
IR
Acetyl-CoA, ATP
Figure 14.1 Derailment of glucose and amino acid metabolism in diabetes. a: Glycol-
ysis and gluconeogenesis; b: Glycogen metabolism; c: Protein degradation in muscle.
See text for details.
192 14 Diabetes mellitus
a) Epinephrine Insulin
β-AR IR
AC
Phosphodiesterase 3
Protein kinase A
Hormone-sensitive lipase
Glucose Pyruvate
activity of the blood and interstitial fluids drains water from the cells, which
hampers cell function.
5. Some patients report a recent flu-like infection, sometimes with chest
pain or even myocarditis (inflammation of the heart muscle).
a)
T T
b)
T cell
receptor
viral cellular
peptide peptide
HLA high-risk HLA
molecule molecule
c)
Amount
excreted
Figure 14.4 Glucose secretion in the urine in diabetes. a: The nephron and its
function. Blood plasma is filtrated in the glomerulus (top), and the filtrate passed
through the tubuli before excretion. Most of the water and many solutes, including
glucose and amino acids, are reclaimed by active transport in the tubuli. b: The
maximum rate of glucose reuptake is limited by the number of transporter molecules.
It is saturated at approximately 10 mM glucose.
The fundamental thing, of course, is to replace the lacking insulin. An acutely ill,
possibly comatose patient will require additional initial measures. Intravenous
infusion therapy is performed, with the following goals:
1. Replacement of fluid,
2. Adjustment of blood pH and electrolytes (potassium, sodium, calcium),
which are commonly derailed by the disturbed kidney function,
3. Rapid adjustment of insulin dosage, guided by frequent monitoring of
14.8 Long-term complications of diabetes mellitus 197
blood glucose.
tions do not require insulin to take up glucose. In these tissues, the intracellular level of glucose
will be higher than in the insulin-dependent ones.
198 14 Diabetes mellitus
before a meal, the time profile of insulin becoming available in the circulation
approximates the duration of the physiological postprandial peak.
The problem that remains then is to keep the level of insulin sufficiently
high in the periods between peaks. To this end, the rate of capillary uptake
must be slowed down even further. Preparations that have been conditioned for
delayed uptake are referred to as long-acting or “basal” insulins, as opposed to
native insulin and other short-acting “bolus” insulins. A combination of basal
and bolus insulins, and optionally intermediate-acting ones, can then be used
to mimic the overall physiological insulin secretion profile. The rate of insulin
monomer release is influenced by a multitude of effects, which can be exploited
in the preparation of long- and intermediate-acting insulins:
1. Insulin is negatively charged at neutral pH, which will cause electrostatic
mutual repulsion of the monomers, promoting dissociation. Adjusting the pH
to lower values will reduce repulsion and promote aggregation.
2. Positively charged additives such as zinc and, more strongly, protamine—
a small, positively charged nuclear protein obtained from the testes of rainbow
trout—will associate with insulin and promote its aggregation.
3. Insulin crystals dissolve more slowly than amorphous aggregates.
4. Addition of two positively charged arginine residues to the carboxy-ter-
minus of the B chain, in combination with substitution of residue asparagine
A21 by glycine, yields “glargine”, a derivative with stable, slow release kinetics.
5. Covalent linkage of a fatty acyl residue to lysine 29 yields “insulin de-
temir”, which may form micellar aggregates or bind to other proteins, causing
slower uptake into and slower clearance from the blood.
The exact mixture and dosage of slow- and fast-acting insulins has to be
adjusted empirically with each patient. Traditionally, when developing an indi-
vidual treatment plan, emphasis was placed on minimizing the number of daily
insulin injections. One limitation of this approach is that the patient needs to
carefully synchronize his meals with his insulin application schedule. Moreover,
the blood glucose level will often not be as tightly controlled as is desirable in
order to minimize the induction of diabetic long-term complications.
Tighter glucose control is the purpose of intensive insulin therapy, in which
frequent measurements of blood glucose are used to guide the likewise more
frequent applications of insulin. One risk inherent in this approach is that
hypoglycemia may result when a dose of insulin is applied before the previous
ones have been fully taken up into the circulation. In order to minimize this
risk, it is desirable to accelerate the capillary uptake beyond the rate achievable
with native insulin. Several mutant insulins have been created that aggregate
less readily than wild type insulin and therefore undergo faster capillary up-
take. Insuline lispro, in which amino acid residues proline B28 and lysine B29
are switched, and insulin aspart, which contains a mutant aspartate residue
at position B28, are in clinical use and reportedly offer a reduced risk of hy-
14.9 Glucose assays 199
Capillary wall
Figure 14.5 Aggregation and capillary wall penetration of insulin. Zinc ions and high
insulin concentration promote the formation of hexamers, which don’t permeate across
the capillary walls. The aggregation equilibrium can be shifted either way by various
point mutations, which is exploited in the preparation of both fast- and slow-acting
insulins (see text for details).
poglycemia. Figure 14.6 illustrates how these changes reduce the stability of
insulin aggregates.
The idea of just-in-time application of insulin leads logically to insulin
pumps, which can release insulin continuously, much like the pancreatic islets.
Ideally, the flow rate would be automatically controlled without any required
user intervention by continuous measurement of the blood glucose level. To
avoid undulations in the feedback loop, the delay between the subcutaneous re-
lease by the pump and the availability of insulin in the circulation should be as
small as possible; therefore, insulin preparations with minimized aggregation
will again be preferable.
Other insulin delivery methods have also been developed. Inhalable insulin
was available on the market for a short while. However, due to the concerns
about the long term effects of insulin on lungs and the accuracy in the dosage,
demand was lower than expected, and the product was terminated.
glucose + O2 ------→
- gluconate + H2 O2 glucose oxidase
H2 O2 + colorless pre-dye ------→
- H2 O + dye peroxidase
In this coupled assay, the amount of dye formed in the second step is propor-
tional to the glucose consumed in the first step. (Note that glucose oxidase is
isolated from microbes but does not have a role in human metabolism).
200 14 Diabetes mellitus
Insulin dimer 1
Insulin dimer 3
Insulin dimer 2
Val 3 Pro 28
Glu 21
Val 12
Tyr 16
Figure 14.6 Structure of the insulin hexamer. The hexamer is composed of three
dimers and stabilized by two centrally placed zinc ions. The two monomers of each
dimer are highlighted in dark and light shades, respectively. In each dimer, the proline
residue at position B29 of one monomer interacts with a patch of hydrophobic residues
(valine B3, valine B12 and tyrosine B16) of the other monomer. In insulin lispro, proline
B28 is replaced with lysine, which destabilizes the interaction of the two monomers. In
insuline aspart, aspartic acid replaces proline B28, which also breaks the hydrophobic
interaction and additionally creates electrostatic repulsion with glutamate B21 of the
other monomer.
Glucose−CH− - Glucose−CH−
−O + H2 N−hemoglobin ------→ −N−hemoglobin + H2 O
on glucose, it will pass out. Now, if you find a known diabetic in a comatose
state, what do you do – give him glucose or insulin? Since the hypoglycemic
coma is more immediately life-threatening than the hyperglycemic one, you
must always use glucose first. Only if that doesn’t help should you try insulin.3
3 This scenario is probably quite hypothetical nowadays, since fast and accurate glucose meters
are now widely available. However, it used to be much more realistic in the days when glucose
assays could only be done in the lab.