BIO 202 Biochemistry II

by
Seyhun YURDUGÜL

Lecture 5
The Citric Acid(Tricarboxylic
Acid/Krebs) Cycle
 Outline
• Introduction
• Reactions
• Energy balance
• Regulation and linkage within the
intermediary metabolism
 The Citric Acid Cycle

• also called the tricarboxylic acid (TCA) cycle;

• and the Krebs cycle.
• the final common catabolic pathway for the
oxidation of fuel molecules.
• Two carbons enter the citric acid cycle as acetyl
CoA;
• and two carbons leave as CO2.
 The Citric Acid Cycle
• In the course of the cycle, four oxidation-
reduction reactions take place;
• to yield reduction potential in the form of
three molecules of NADH;
• and one molecule of FADH2.
• A high energy phosphate bond (GTP) is
also formed.
Linkage to other metabolic
pathways
Since cycle intermediates can be
incorporated into both anabolic
and catabolic pathways, the cycle
is really amphibolic, not just
catabolic
 Anaplerotic pathway
• If an intermediate in any pathway:
• replenished by another pathway’s
intermediate:
• A typical example of anaplerotic pathway.
• E.g.malate in the mitochondrial matrix
replenishes pyruvate.
• Anaplerotic: of Greek origin, meaning to fill
up.
 The first reaction:
Pyruvate to Acetyl Co-A

• In reality, this reaction:

• not really in the Krebs Cycle,
• but since it is the first reaction that occurs
in the mitochondrion;
• and it leads directly into the cycle,
• it is usually included in the discussions of
the cycle.
 The first reaction:
Pyruvate to Acetyl Co-A
• In this reaction, pyruvate, a three carbon
molecule that is generated in glycolysis
and;
• in the metabolism of some amino acids,
• is decarboxylated (a carboxyl group is
removed) to the two carbon acetate
 The first reaction:
Pyruvate to Acetyl Co-A
• The carboxyl group:
• released as carbon dioxide.
• catalyzed by the enzyme pyruvate
dehydrogenase.
• This reaction is also an oxidation;
• as 2 electrons are removed from pyruvate
during the reaction
Pyruvate Dehydrogenase Complex
Structure of the thiamin diphosphate dependent enzyme
pyruvate decarboxylase-brewer's yeast Saccharomyces
cerevisiae uvarum strain
Pyruvate Dehydrogenase Complex
The lipoyl E2 domain of complex which serves as an acyltransferase.

                                                                 

From: Azotobacter vinelandii

 The first reaction:
Pyruvate to Acetyl Co-A

• The two electrons:

• accepted by NAD and results in the
formation of NADH.
• This oxidation is very exergonic (ΔG = -7.5
kcal/mole).
 The first reaction:
Pyruvate to Acetyl Co-A
• Some of the energy released from this
reaction: transferred with the electrons to
NADH;
• and some: used to energize acetate by
adding coenzyme A to acetate;
• forming acetyl CoA, the actual product of
the reaction
 Note about this reaction:

• Pyruvate; the product of glycolysis, comes

from glucose.
• It may also come from some amino acids.
• reaction occurs in the mitochondrion.
 Note about this reaction:
• One carbon:
• removed from pyruvate in the form of
carbon dioxide (note the yellow carbon in
the figure of slide 10).
• This leaves just two carbons remaining
from pyruvate.
 Note about this reaction:
• The addition of the coenzyme A to the
acetate (see red in the above figure of
slide 8);
• acts to conserve the energy;
• released from the reaction and to energize
the acetate.
 Step 2: Condensation
Acetyl Co-A to Citrate via
Oxaloacetate

• In step 1 of the Krebs cycle,

• the two-carbon compound, acetyl-S-CoA,
• participates in a condensation reaction
with the four-carbon compound,
• oxaloacetate, to produce citrate:
 Step 2: Condensation
Acetyl Co-A to Citrate via
Oxaloacetate
• moderately exergonic reaction.
• Thermodynamically, the equilibrium is in
favor of the products.
• considered to be the first committed step
of the Krebs cycle
 Step 2: Condensation
Acetyl Co-A to Citrate via
Oxaloacetate
• Being the first committed step,

• this is a likely step to have some kind of

regulatory control mechanism.

• which will effectively regulate the entire cycle.

 Step 2: Condensation
Acetyl Co-A to Citrate via
Oxaloacetate
• The Krebs cycle is also known as the citric
acid cycle.
• Citrate is a tricarboxylic acid,
• and the Krebs cycle is also known as the
tricarboxylic acid (or TCA) cycle
 In other words: Oxaloacetate +
Acetyl CoA to Citrate

• Enzyme: Citrate synthase

• Reaction: Condensation
• Oxaloacetate condenses with acetyl CoA to
form citryl CoA.
• Then citryl CoA is hydrolyzed to citrate and
CoA.
• Prosthetic group: No
 Step 3. Isomerization of
Citrate

• a decarboxylation reaction.
• usually involve α- (or ß-) keto acids
• hydroxyl group of citrate can be oxidized
to yield a keto group,
• but to form an α-keto acid;
• it needs to be adjacent to one of the
terminal carboxyl groups
 Step 3. Isomerization of
Citrate
• involves moving the hydroxyl group in the citrate
molecule;
• so that α-keto acid is formed.
• involves a sequential dehydration and hydration reaction,
• to form the D-Isocitrate isomer;
• with the hydroxyl group now in the desired α- location,
• with cis-Aconitase as the intermediate
• A single enzyme, Aconitase, performs this two-step
process:
Aconitase
Aconitase (E.C.4.2.1.3) in the activated (4Fe-4S)
cluster form.
Step 3. Isomerization of Citrate
 Citrate to cis-Aconitate

• Enzyme: Aconitase
• Reaction: Dehydration
Citrate: isomerized to isocitrate by this first
dehydration;
• and yields cis-aconitate as an intermediate.
• Prosthetic group: Fe-S
 cis-Aconitate to Isocitrate

• Enzyme: Aconitase
• Reaction: Hydration
Hydration of cis-aconitate gives the
interchange of H atom and OH group from
the step 2.
• Prosthetic group: Fe-S
 cis-Aconitate to Isocitrate
• This reaction is endergonic,
• so the equilibrium is in favor of the
reactants;
• and not the desired product.
• However, the exergonic character of the
next reaction in the cycle:
• helps shift the equilibrium of this reaction
towards the right.
 cis-Aconitate to Isocitrate
• Two asymmetric centers in the D-Isocitrate
molecule:
• Each can adopt either the L- or D-form,
• thus there are 4 possible isomers of this
molecule
 Aconitase enzyme

• only produces the single form of Isocitrate

(D-Isocitrate).
• a stereospecific enzyme
 Step 4: Generation of CO2 by
an NAD+ linked enzyme

• The Krebs cycle contains two oxidative

decarboxylation steps;
• this is the first one
• The reaction is catalyzed by the enzyme
Isocitrate dehydrogenase
 Step 4
Isocitrate to α-Ketoglutarate
• Enzyme: Isocitrate dehydrogenase
• Reaction: Oxidative decarboxylation
• Dehydrogenation of isocitrate occurs;
• and yields oxalosuccinate as an
intermediate.
• Then CO2 leaves to have alpha-ketoglutarate.
• This reaction gives NADH.
• Prosthetic group: No
Isocitrate Dehydrogenase
Isocitrate Dehydrogenase (E.C.1.1.1.42) with NADP
 Step 5:
alpha-Ketoglutarate to Succinyl
CoA

• Enzyme: alpha-Ketoglutarate dehydrogenase

complex
• Reaction: Oxidative decarboxylation
• almost as same as the reaction of the oxidative
decarboxylation of pyruvate to acetyl CoA;
• by pyruvate dehydrogenase complex.
• reaction gives one NADH.
• Prosthetic group: Lipoic acid, FAD, TPP
 Step 6: Succinyl CoA to
Succinate

• Enzyme: Succinyl CoA synthetase

• Reaction: Substrate-level
phosphorylation
• The thioester bond of succinyl and
CoA:
• an energy rich bond.
 Step 6: Succinyl CoA to
Succinate
• only this step gives a high-energy
phosphate compound,
• GTP from the couple reactions of the
thioester bond cleavage
• and the phosphorylation of GDP.
• Prosthetic group:No
Succinyl-CoA Synthetase
Succinyl-CoA Synthetase (E.C.6.2.1.5) with coenzyme A
 Step 7: Succinate to Fumarate

• Enzyme: Succinate dehydrogenase

• Reaction: Oxidation
• The two hydrogens of succinate leave
to an acceptor, FAD.
• Then this reaction yields fumarate and
FADH2.
• Prosthetic group: FAD & Fe-S
 Succinate to fumarate
• When succinate is brought into;
• or generated in the mitochondria matrix in
sufficient quantity,
• succinate molecules bind the enzyme
complex called succinate dehydrogenase.
 Succinate to fumarate
• succinate dehydrogenase complex:
• also known as complex II of the electron
transport system,
• thus the oxidation of succinate to fumarate
is the only Krebs reaction;
 Succinate to fumarate
• that takes place on the inner membrane
itself,
• the other reactions: catalyzed by soluble
enzymes.
• The energy carrier flavin adenine
dinucleotide (FAD):
• also a part of the succinate
dehydrogenase complex
 Succinate to fumarate
• as the enzyme and FAD:
• both part of the same complex,
• the only step needed to initiate succinate
oxidation:
• the binding of succinate to the enzyme.
• mitochondria succinate supported respiration
can usually be accomplished,
• as long as fragments of the inner membrane
remain
 Step 8: Fumarate to Malate via
Fumarase
• that catalyzes the hydration;
• addition of H2O across a double bond;
• of Fumarate to L-malate
Fumarase
Fumarase with bound malate.
 Malate to Oxaloacetate

• Enzyme: Malate dehydrogenase

• Reaction: Oxidation
• Malate: dehydrogenated to form
oxaloacetate.
• The hydrogen acceptor: NAD+.
• So this reaction yields NADH.
• Prosthetic group: No
Malate Dehydrogenase
Malate Dehydrogenase (E.C.1.1.1.37) a complex of the
apoenzyme and citrate
 Energy Balance in Krebs
• Glycolysis:
• 4 ATP molecules are produced ,
• but 2 ATP's are used in the process;
• so the total balance: 2 ATP's.
• In this stage 2 NAD+ 's become NADH's.
 Energy Balance in Krebs
• Krebs Cycle
BEFORE the cycle:
• 2 NADH's are created in the creation of Acetyl
Coenzyme A.

• IN the cycle 2 GTP's are created:

• 6 NADH's are created,

• 2 FADH2's too.
 Energy Balance in Krebs
• Electron Chain:
• Every NADH produces 3 ATP's.
• For 10 NADH's: 30 ATP's are created.
Every FADH2 produces 2 ATP's.
• We have 2 FADH2's:
• 4 ATP's are created.
 Total Balance
• 2 ATP + 2GTP +34 ATP:
• 38 ATP
• (Glycolysis + Krebs Cycle + Electron
Chain) (Total)
 Regulation

• Many of the enzymes in the TCA cycle:

• regulated by negative feedback from ATP
• when the energy charge of the cell is high.
 Regulation
• Such enzymes include the pyruvate
dehydrogenase complex;
• that synthesises the acetyl-CoA needed
for the first reaction of the TCA cycle.
 Regulation
• Also the enzymes:
• citrate synthase,
• isocitrate dehydrogenase;
• and alpha-ketoglutarate dehydrogenase,
• that regulate the first three steps of the
TCA cycle,
• are inhibited by high concentrations of
ATP.
 Regulation
• This regulation ensures that the TCA
cycle; will not oxidize excessive amount of
pyruvate;
• and acetyl-CoA when ATP in the cell is
plentiful.
• This type of negative regulation by ATP:
• by an allosteric mechanism
 Regulation
• Several enzymes:
• also negatively regulated;
• when the level of reducing equivalents in a
cell are high (high ratio of NADH/NAD+).
 This mechanism for regulation:

• due to substrate inhibition by NADH of the

enzymes;
• that use NAD+ as a substrate.
• includes both the entry point enzymes;
• pyruvate dehydrogenase;
• and citrate synthase.
 The Electron Transport System
of Mitochondria
• Embedded in the inner membrane are proteins;
• and complexes of molecules that are involved in
the process;
• called electron transport.
• The electron transport system (ETS), as it is
called,
• accepts energy from carriers in the matrix;
• and stores it to a form that can be used to
phosphorylate ADP.
 Two energy carriers:

• known to donate energy to the ETS,

• namely nicotine adenine dinucleotide
(NAD+);
• and flavin adenine dinucleotide (FAD).
 Two energy carriers:
• Reduced NAD+:
• carries energy to complex I (NADH-
Coenzyme Q Reductase) of the electron
transport chain.
• FAD+: a bound part of the succinate
dehydrogenase complex (complex II).
 LITERATURE CITED
• Devlin,T.M. Textbook of Biochemistry with
Clinical Correlations,Fifth Edition,Wiley-Liss
Publications,New York, USA, 2002.
• Lehninger, A. Principles of Biochemistry, Second
edition, Worth Publishers Co., New York, USA,
1993.
• Matthews, C.K. and van Holde, K.E.,
Biochemistry, Second edition, Benjamin /
Cummings Publishing Company Inc., San
Francisco, 1996.