Enzyme Regulation

Factors influence enzymatic activity

Genetic regulation of enzyme synthesis
and decay determines the amount of
enzyme present at any moment
Enzyme activity can be regulated
allosterically
Enzyme activity can be regulated through
reversible covalent modification
Zymogens (proteolytic cleavage)
Isozymes (multiple forms of isoenzymes)
Binding with modulator proteins
 Enzyme regulation by reversible covalent
modification

Phosphorylation is a highly effective means of controlling the

activity of proteins for several reasons:
1) ATP is the cellular energy currency
2) The free energy of phosphorylation is large
3) A phosphoryl group adds two negative charges to a modified
protein
4) A phosphoryl group can form three or more hydrogen bonds
Isozymes are enzymes with slightly
different subunits
Isozymes of lactate dehydrogenase
n Rat LDH isozymes have different n Rat LDHs have different
expression profile in different tissues composition in different
developmental stages

M isozyme H isozyme

H isozyme is highly expression in heart muscle and the M isozyme is

expressed in skeletal muscle.
The H4 isozyme has a higher affinity for substrate than does the M4 isozyme.
The M4 isozyme functions optimally in the anaerobic environment of
hard-working muscle, whereas the H4 does so in the aerobic
environment of heart muscle.
 Some enzymes and proteins are regulated by
proteolytic cleavage
An inactive precursor called a zymogen (proenzyme)
or a proprotein is cleaved to become the active form.
Specific cleavage causes conformation changes that
expose the active site.
Because this type of activation is irreversible, other
mechanism are needed to inactivate these enzymes.
For example, proteases are inactivated by binding
with inhibitors very tightly to the active site.
An energy source such as ATP is not needed for
cleavage. Proteins located outside cells can still be
activated.
 Enzymes and proteins activated by specific
proteolysis in biological systems
Some protein hormones (an 86-residue
proinsulin to 51-residue insulin)
The digestive enzymes
Blood clotting
The fibrous protein collagen (derived
from procollagen)
Many developmental processes
(collagenase are derived from
procollagenase)
Programmed cell death, or apoptosis
(caspases are derived from
procaspases)
The proteolytic activation of chymotrypsinogen
Activation of zymogens by proteolytic cleavage

Three polypeptide chains (A, B,

and C) of chymotrypsin are
linked by disulfide bonds
Protonated Ile16 forms salt-
bridge with Asp194
Chymotrypsinogen Chymotrypsin

D194 D194
H57
S195
D102 D102 H57 S195
I16

I16 Distance between D194

and I16: 2.62
Distance between D194
and I16: 18.28
Many enzymes are activated by specific
proteolytic cleavage

Secreted by duodenum cells

D-D-D-D-K-I
 The cascade of activation steps leading to
blood clotting

The intrinsic and

extrinsic pathways
converge at factor X,
and the final common
pathway involves the
activation of thrombin
and its conversion of
fibrinogen into fibrin,
which aggregates into
ordered filamentous
arrays that crosslink to
form the clot.
 Blood clots are formed by a cascade of
zymogen activations
Very small amounts of the initial
factors suffice to trigger the
cascade, ensuring a rapid
response
Hemophilia A: Factor VIII
(antihemophilic factor) is missing
or has markedly reduced activity
Tissue factor is an integral
membrane glycoprotein.
Factors in blue are not enzymes
The active forms in yellow are
designated with an subscript a.
Factors activated by thrombin are
designated with an asterisk ( )
Positive feedback accelerates
reactions after a threshold has
been reached
 Prothrombin is activated by a vitamin K-
dependent proteolytic cleavage

1st : R-274/T-275 2nd : R-323/I-324

These Gla/Kringle/Kringle domains work in concert to keep prothrombin in

an inactive form and to target it to appropriate sites for its activation by
factor Xa (a serine protease) and factor Va (a stimulatory protein).
Activation is begun by proteolytic cleavage of the bond between R-274 and
T-275. Cleavage of the bond between R-323 and I-324 yields active
thrombin.
The first 10 glutamate residues in the N-terminal region of prothrombin
are carboxylated to g-carboxyglutamate by a vitamin K-dependent
enzyme system. This process turns a weak chelator (glutamate) of Ca2+
into a much stronger chelator (g-carboxyglutamate).
Binding of Ca2+ anchors prothrombin to phospholipid membranes
derived from blood platelets after injury, where Xa and Va catalyze its
conversion into thrombin.
The calcium-binding domain is removed during activation, freeing the
Calcium-binding
thrombin from the membrane so that it can cleave fibrinogen and other
region targets.
of prothrombin
General features of allosteric regulation

Allosteric: Action at "another site

Enzymes situated at key steps in
metabolic pathways are modulated by
allosteric regulation
Allosteric effectors may be feedforward
activators or feedback inhibitors
Kinetics are sigmoidal ("S-shaped")
Allosteric kinetics are sigmoidal

The dotted line represents the hyperbolic

characteristic of normal Michaelis-Menten kinetics
 Allosteric regulation is based on two
conformational states for a protein

Allosteric proteins can exist in two states: R (relaxed)

and T (tense)
Allosteric activator (A) and inhibitor (I) binding to R and
T, respectively.
 Aspartate transcarbamoylase is allosterically
inhibited by the end product of its pathway

CTP and ATP are not

the substrates of
ATCase
ATP can recover the
allosteric inhibition
triggered by CTP

The feedback inhibition of ATCase by CTP (the end product

of the pathway) ensures that N-carbamoylaspartate and
subsequent intermediates in the pathway are not
needlessly formed when pyrimidines are abundant.
 ATCase displays
CTP inhibits ATCase
sigmoidal kinetics

n A sigmoidal curve indicates that p CTP has no structural similarity to

binding of substrate to one active site reactants or products.
increases the activity at the other sites. p Thus, CTP must bind to a site
n Allosterically regulated enzymes do distinct from the active site at
not follow Michaelis-Menten kinetics. which substrates bind.
Structure of ATCase, c6r6
r2

r2
c3 r2
c3

c3
Basis for the sigmoidal curve
Homotropic allosteric effect Heterotropic allosteric effect

Homotropic allosteric modulator

CTP stabilizes the T state

Heterotropic allosteric modulator
Effects of CTP and ATP on ATCase kinetics
p CTP stabilizes the T state, making it p ATP stabilizes the R state, making
more difficult for substrate binding it more easier for substrate binding

p In the absence of any substrate or regulators, the T

state is favored by a factor of approximately 200.

Tstate
L=
Rstate
 Protein kinase
Protein kinases phosphorylate Ser, Thr, and Tyr
residues in target proteins
Kinases typically recognize specific amino acid
sequences in their targets
All kinases share a common catalytic mechanism
based on a conserved core kinase domain of
about 260 residues
Kinases are often regulated by intrasteric
control, in which a regulatory subunit (or domain)
has a pseudosubstrate sequence that mimics
the target sequence without the phosphorylatable
residue
Classification of protein kinases
Regulation of protein kinase A by cAMP

Cyclic AMP-dependent protein kinase A, also known as protein

kinase A (PKA)
The pseudosubstrate sequence (Arg-Arg-Gly-Ala-Ile) of regulatory
subunit (R) occupies the active site of catalytic subunit (C), thereby
preventing the entry of protein substrates.
Cyclic AMP activates protein kinase A by binding to R chains that
allosterically moves the pseudosubstrate sequences out of C chains
and subsequently alters the quaternary structure.
Phosphorylation is not the only form of covalent
modification that regulates protein function
 Enzyme activity can be regulated through
reversible phosphorylation
Phosphorylation/dephosphorylation is the most
prominent form of covalent modification in cellular
regulation
Phosphorylation is accomplished by protein
kinases. Each protein kinase targets specific
proteins for phosphorylation
Phosphoprotein phosphatases catalyze the
reverse reaction dephosphorylation
Sometimes, kinases and phosphatases
themselves are also the targets regulated by other
kinases and phosphatases.
Glycogen phosphorylase is controlled by both
allosteric regulation and covalent modification

Glycogen phosphorylase (GP) is a dimer of identical

842 residue subunits, each with pyridoxal phosphate
(PLP) covalently linked
Each subunit contains an active site (at the center of
the subunit) and an allosteric effector site near the
subunit interface
GP cleaves glucose units from nonreducing ends of
glycogen into glucose-1-phosphate through a
phosphorolysis reaction
A regulatory phosphorylation site is located at
Ser14 on each subunit
 The mechanism of covalent modification and
allosteric regulation of glycogen phosphorylase
 Regulation of muscle glycogen phosphorylase
activity by multiple mechanisms
(Glucose)n + Pi
(glucose)n 1 + glucose 1-
phosphate

Adenylyl
cyclase
GP

Ser14 Ser14 Ser14 Ser14 The activity of GP in
muscle is regulated by
covalent modification
(phosphorylation),
allosteric regulation,
and a regulatory
cascade sensitive to
hormonal status that
acts on the enzymes
involved in
Adenylyl phosphorylation and
cyclase dephosphorylation.
 The activity of glycogen phosphorylase is
regulated allosterically
Muscle GP shows cooperativity in substrate binding
ATP and glucose-6-P are allosteric inhibitors of GP
AMP is an allosteric activator of GP
When ATP and glucose-6-P are abundant, glycogen
breakdown is inhibited (T state); When cellular energy
reserves are low (i.e., high [AMP] and low [ATP] and [G-6-
P]) glycogen catabolism is stimulated (R state)
 Glycogen phosphoryase is activated by a
cascade of reactions
Cyclic AMP is the intracellular
agent of extracellular hormones -
thus a second messenger

The hormone-activated enzymatic cascade that

leads to activation of glycogen phosphorylase.
The adenylyl cyclase reaction
cAMP is a second messenger
Hormone binding to its receptor
stimulates the activation of the
GTP-binding protein (G protein),
leading to release G(GTP)
Binding of G(GTP) to adenylyl
cyclase stimulates cAMP
synthesis
Adenylyl cyclase and the
hormone receptor are integral
membrane proteins
G and G are membrane-
anchored proteins
O2-binding curves for hemoglobin and
myoglobin

Hb must be able to bind oxygen in the lungs

Hb must be able to release oxygen in capillaries
If Hb behaved like Mb, very little oxygen would be released in capillaries
The sigmoid, cooperative oxygen-binding curve of Hb makes its
physiological actions possible
 For their studies of the structures of
globular proteins
1962 Perutz and Kendrew won the
Nobel Prize in 1962 for solving
the structures of hemoglobin
(Perutz) and myoglobin
(Kendrew). This is the same
year that Watson, Crick, and
Wilkins won for the structure of
DNA. Recall that Watson &
Crick were working in the
Perutz lab at the time of their
discovery and Crick was
actually working on the
structure of hemoglobin as part
of his Ph.D. thesis
Max Perutz, 1914-2002 (left) John kendrew, 1917-1997 (right)
Myoglobin was the first protein for which the
three-dimensional structure was determined
Revealed by John Kendrew et al. in 1950s

(Sperm whale)

Biochemistry, 7th edition (2010), Berg, Tymoczko and Stryer

 Hemoglobin and Myoglobin
Hemoglobin (Hb) and myoglobin (Mb) are
oxygen- transport and oxygen-storage proteins,
respectively
Mb is monomeric (153 aa, 17.2 kDa) ; Hb is
tetrameric
Hb is an 22 tetramer (two chains of 141
residues, 2 chains of 146 residues)
Mb is composed of 8 helices
Hb chain has 7 helices, Hb chain has
8 helices
Interior of Mb contains almost all hydrophobic
amino acids
Tertiary structures of myoglobin, -
hemoglobin and -hemoglobin

-hemoglobin (blue)
-hemoglobin (purple)
Myoglobin (green)
The amino acid sequences of whale myoglobin and the
and chains of human hemoglobin

Lehninger Principles of biochemistry, 5th edition

Cooperativity enhances oxygen delivery by Hb

Hyperbolic

S
shaped

p Hemoglobin is more sensitive to the small differences

in O2 concentration between the tissues and the lungs
Spectroscopic detection of oxygen binding
to myoglobin
pThe heme group is a strong chromophore that
absorbs both in ultraviolet and visible range
pSoret band (named after its discoverer Jacques-
Louis Soret): a very strong absorption band in the
blue wavelength region of the optical absorption
spectrum of a heme protein
pFerrous form (Fe2+ ) without oxygen has an intense
Soret band at 429 nm
pOxygen binding alters hemes electronic properties,
and shifts the position of the Soret band to 414 nm
pDeoxymyoglobin (in venous blood) appears purplish
in color and oxymyoglobin (in arterial blood) is red
Mb and Hb use heme to bind Fe2+

p Heme is formed when protoporphyrin IX binds Fe2+

 Porphyrin & the heme group

Simplest porphyrin

p Porphyrin coordinated
to iron: Heme
p Porphyrin coordinated
to magnesium:
Chlorophyll
p The one-carbon-shorter
analogue corrole
coordinated to a cobalt Myoglobin/Hemoglobin/Cytochrome
: Vitamin B12
Biochemistry, 7th edition (2010), Berg, Tymoczko and Stryer
The key residues around the Fe2+ heme
upon oxygen binding
His-64
Distal
histidine

The O2 is
H-bond tilted

Proximal
His-93 histidine
 Oxygen binding changes the position
of the iron ion
p Iron ion moves into the plane of the heme on oxygenation
predicted by Linus Pauling in 1936
O2

Biochemistry, 7th edition (2010), Berg, Tymoczko and Stryer

 Conformational changes in a hemoglobin
chain induced by oxygenation
Oxygen binding to Fe pulls the His F8 toward ring plane
O2

p For Mb, this small change has little consequence.

p But a similar change in Hb initiates a series of conformational changes that
are transmitted to adjacent subunits.
 Changes in conformation near heme on O2
binding to deoxyhemoglobin
Lehninger Principles of biochemistry, 5th edition
 Fe2+ movement (upon O2 binding) by less than
0.04 nm induces the conformational change in Hb

p In deoxy-Hb, the iron atom lies out of the heme

plane by about 0.06 nm
p Upon O2 binding, the Fe2+ atom moves about
0.039 nm closer to the plane of the heme
p As Fe2+ moves, it drags His F8 and the F helix
with it
p This change is transmitted to the subunit
interfaces, where conformation changes lead
to the rupture of salt bridges
 Oxygen binding to Hb results in a 15
rotation of one pair relative to the other
 The T R transition

Deoxyhemoglobin Oxyhemoglobin
T: tense (low-affinity state) R: relaxed (high-affinity state)
Lehninger Principles of biochemistry, 5th edition
Some ion pairs between 2 and 1, and within 1
that stabilize the T state of deoxyhemoglobin

1
2

Lehninger Principles of biochemistry, 5th edition

 Some ion pairs that stabilize the T state of
deoxyhemoglobin
*

**

**
*

p Some ion pairs (salt bridges) between subunits are

disrupted while oxygen binding to hemoglobin
Lehninger Principles of biochemistry, 5th edition
 T-to-R transition
p A combination of the binding curves of T state
and R state would be observed
Oxygen Binding by pure hemoglobin compared
with hemoglobin in red blood cells

p Oxygen binding to Hb is
regulated by 2,3-
bisphosphoglycerate
(2,3BPG)
p 2,3BPG is an allosteric
effector of Hb
p 2,3BPG lowers the
affinity of deoxyHb for
oxygen (raises the P50 of
Hb from ~12 to ~26 torr)
Effect of BPG on oxygen binding to deoxyHb

2,3BPG lowers
the affinity of
deoxyHb for
oxygen

p The BPG concentration in

normal human blood is about
5 mM at sea level and about
8 mM at high altitudes

Lehninger Principles of biochemistry, 5th edition

Binding of 2,3BPG to the central cavity of Hb
stabilizes the DeoxyHb form

+
+
+
+ +
+ +
+

p Negatively charged 2,3BPG interacts with 8 positive charges

in the cavity of deoxyhemoglobin: 2 Lys, 4 His, 2 N-termini
 Fetal red blood cells have a higher oxygen affinity
than do maternal red blood cells

p The fetus synthesizes subunits rather than subunits, forming 22

hemoglobins. His-143 in chains, part of the BPG-binding site, is
substituted to a Ser residue in subunits. This change removes
two positive charges from the BPG-binding site (one from each
chain). Consequently, 22 has a much lower affinity for BPG than
normal hemoglobin, and a corresponding higher affinity for oxygen.
Effect of pH on the oxygen affinity of Hb
p Lowering the pH decreases the affinity of Hb for oxygen

p The effect of pH and CO2

concentration on the
binding and release of O2
by hemoglobin is called
Bohr effect, first described
in 1904 by Christian Bohr
(the father of Neils Bohr, the
atomic physicist)
p The pH difference between
lungs and metabolic tissues
increases the O2 transfer
efficiency of Hb.
CO2 decreases the affinity of Hb for oxygen even
beyond the effect due to a decrease in pH

CO2 (lowering the

pH) makes Hb
tends to release
O2 in tissues
 Chemical basis of the Bohr effect
p As the pH drops, the side chain of His-146 becomes
protonated, the salt bridge with Asp-94 forms, and the T state
is stabilized, leading to a greater tendency for oxygen to be
released.
p Low pH makes T state more
stabilized through the
formation of His146-Asp94
salt bridge in subunits
 Binding of carbon monoxide to Hb
p CO has similar size and shape to O2; it can fit to the
same binding site.
p Heme binds CO 20,000 times better than O2
because the C in CO has a filled lone electron pair
that can be donated to vacant d-orbitals on the Fe2+.
p Protein pocket decreases affinity for CO, but is still
binds about 250 times better than oxygen.
p CO is highly toxic as it competes with oxygen. CO
blocks the function of myoglobin, hemoglobin, and
mitochondrial cytochromes that are involved in
oxidative phosphorylation.
Binding of carbon monoxide to Hb

n Fetal 22 Hb
has a higher
affinity for CO
than adult Hb

Lehninger Principles of biochemistry, 5th edition

 Sickle-cell
Sickled red blood cells
hemoglobin fibers
 Sickle-cell anemia results from the aggregation
of mutated deoxyhemoglobin molecules (HbS)
Demonstrated by Vernon Ingram in 1956
GAA GTA
Glu Val

p This was the first time a researcher demonstrated that a single amino acid
exchange in a protein can cause a disease or disorder. As a result, Vernon
Ingram is sometimes referred to as "The father of Molecular Medicine".
 Normal and sickle-cell hemoglobin

l The altered properties of hemoglobin S

result from a single amino acid
substitution, E6V in two subunits.
l Deoxyhemoglobin S has a
l Valine creates a sticky hydrophobic hydrophobic patch on its surface,
contact point at position 6 of the which causes the molecules to
chain, which is on the outer surface of aggregate into strands that align
the molecule. into insoluble fibers
 Sickle-cell
Sickled red blood cells
hemoglobin fibers
Sickle-cell trait and malaria ()

MyoglobinHemoglobin
Protein tertiary structure (peptide bond, -helix,
hydrophobic core); Protein quaternary structure
Have similar structure, but different a.a. sequences
Nonprotein part (prosthetic group, Fe2+, heme)
The versatile functions of His (binding Fe2+, binding O2,
formation of a salt bridge with Asp, Bohr effect)
Hyperbolic and sigmoidal O2 binding curve
Kd (the [L] at half , ligand affinity)
Soret band (absorbance measurement)
Tense (T) and Relaxed (R) forms
Allosteric regulation (O2, 2,3BPG)
Isoforms (adult 22, infant 22)
CO binding (substrate analog inhibitor)
Single residue mutation leads to conformational
change (1 seq decides 3-D) and Sickle-cell anemia
BIOCHEMISTRY

shihchung@ntu.edu.tw