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Part III => METABOLISM and ENERGY
§3.5 KREBS CYCLE
§3.5a Pyruvate Oxidation
§3.5b Krebs Cycle Reactions
§3.5c Krebs Cycle Regulation
Sources of Acetyl Group of Acetyl-CoA
- Acetyl group of acetyl-CoA can be derived from the
breakdown of:
(1) Carbs (glucose)
 §3.2
(2) Lipids (fatty acids)
 §3.4
(3) Proteins (amino acids)  §3.4
- Under normal physiological conditions, the acetyl
group of acetyl-CoA is largely derived from pyruvate
(the end product of glycolysis)—see §3.2
- Pyruvate is transported into the mitochondrial matrix
by the pyruvate-proton symporter located within the
highly selective inner mitochondrial membrane
(IMM)—the relatively-porous outer mitochondrial
membrane (OMM) harbors large non-selective
channels such as voltage-dependent anion channels
and porins, which enable facilitated diffusion of most
metabolites into the intermembrane space (IMS)
- Within the mitochondrial matrix, oxidation of acetyl
group of acetyl-CoA to CO2 via Krebs cycle generates
free energy that is captured in the form of reduced
NADH/FADH2 and “high-energy” GTP
Section 3.5a:
Pyruvate Oxidation
Synopsis 3.5a
- A major source of acetyl group of acetyl-CoA entering the Krebs cycle is
derived from pyruvate generated from carbohydrates via glycolysis
- Transfer of the aceto group of pyruvate to acetyl-CoA occurs via oxidative
decarboxylation—involving a five-step reaction catalyzed by a multi-enzyme
system collectively referred to as “pyruvate dehydrogenase complex” or PDC
- Each of the five steps catalyzed by PDC, occurring within
the mitochondrial matrix, requires a specific coenzyme:
(1) Thiamine pyrophosphate (TPP)
(2) Lipoamide/Lipoic acid (LPA)
(3) Coenzyme A (CoA)
(4) Flavin adenine dinucleotide (FAD)
(5) Nicotinamide adenine dinucleotide (NAD+)
- Synthesis of acetyl-CoA from pyruvate can be considered
as a “preparatory stage”, or Step 0 of the Krebs cycle
- Overall reaction (irreversible) catalyzed by PDC is:
Pyruvate + CoA + NAD+ ==> Acetyl-CoA + CO2 + NADH
PDC Catalysis: Multienzyme System
- Pyruvate dehydrogenase complex (PDC) is a
multi-enzyme system comprised of
three distinct subenzymes:
(a) Pyruvate dehydrogenase
(E1)
(b) Dihydrolipoamide transacetylase (E2)
(c) Dihydrolipoamide dehydrogenase (E3)
- PDC catalyzes the overall reaction:
Pyruvate + CoA + NAD+
Acetyl-CoA + CO2 + NADH
- This reaction can be subdivided into five
distinct catalytic steps, each mediated by
a specific subenzyme-coenzyme system, as
outlined below:
(1) E1.TPP
(2) E2-LPA
(3) E2.CoA
(4) E3.FAD
(5) E3.NAD+
PDC Catalysis: (Dihydro)lipoamide
Reduction
Oxidation
Lipoamide
Dihydrolipoamide
Lipoic Acid
Lipoic acid is covalently attached via an
amide linkage to a lysine residue in E2
dihydrolipoamide transacetylase—hence
often termed “lipoamide”
Specific roles of the three subenzymes in PDC are:
(1) Pyruvate dehydrogenase (E1)—oxidizes/decarboxylates pyruvate, transferring the
acetyl group to bound TPP cofactor
(2) Dihydrolipoamide/dihydrolipoyl transacetylase (E2)—transfers the acetyl group
from TPP in E1 to bound lipoamide cofactor, and then from lipoamide to CoA
resulting in the reduction of lipoamide to dihydrolipoamide
(3) Dihydrolipoamide/dihydrolipoyl dehydrogenase (E3)—oxidizes dihydrolipoamide
to lipoamide in E2 using FAD and NAD+ as oxidizing agents
PDC Catalysis: Catalytic Steps
LPA
1
2
3
4
5
Lipoamide (LPA)
Cys-Cys
disulfide bridge
PDC Catalysis: (1) E1.TPP
Thiamine Pyrophosphate (TPP)
Pyrimidine
Thiazole
Pyrophosphate
TPP consists of a central thiazole ring
harboring a carbanion flanked between
a pyrimidine ring and pyrophosphate
- E1 pyruvate dehydrogenase (which harbors TPP cofactor) decarboxylates pyruvate
resulting in the formation of hydroxyethyl-TPP.E1 carbanion intermediate
- The reaction is mediated by the nucleophilic attack of TPP carbanion on the carbonyl
atom of the acetyl moiety on pyruvate
- Recall that TPP is also required for the decarboxylation of pyruvate to acetaldehyde
by pyruvate decarboxylase during alcoholic fermentation (see §3.2c)
PDC Catalysis: (2) E2-LPA
Hydroxyethyl-TPP.E1
Lipoamide (LPA)
Lipoamide consists of lipoic acid
covalently linked via an amide
bond to the ε-amino group of a
lysine residue in E2
Hydroxyethyl moiety of hydroxyethyl-TPP.E1 intermediate is transferred to E2
dihydrolipoamide transacetylase harboring a lipoamide cofactor (lipoamide-E2) via two steps:
(1) Hydroxyethyl carbanion in the hydroxyethyl-TPP.E1 complex launches nucleophilic attack
on the lipoamide disulfide in lipoamide-E2
(2) Hydroxyethyl carbanion is subsequently oxidized to an acetyl group with concomitant
reduction of lipoamide resulting in the formation of acetyl-dihydrolipoamide-E2
intermediate and regeneration of active TPP.E1
PDC Catalysis: (3) E2.CoA
Reactive thiol group
Coenzyme A (CoA)
- E2 dihydrolipoamide transacetylase
catalyzes the transfer of acetyl group of
acetyl-dihydrolipoamide-E2 intermediate to
CoA
- This results in the formation of acetyl-CoA
(the Krebs cycle substrate) with concomitant
release of dihydrolipoamide-E2 intermediate
- The subsequent reactions merely act to
regenerate active lipoamide-E2 complex
PDC Catalysis: (4) E3.FAD
Cys-Cys
disulfide bridge
E3(ox).FAD
Dihydrolipoamide-E2
- Oxidized E3 dihydrolipoamide dehydrogenase
[E3(ox).FAD] oxidizes the dihydrolipoamide-E2
intermediate to active lipoamide-E2 complex in
a disulfide exchange reaction
- In so doing, the disulfide linkage of E3 becomes
reduced to thiol groups resulting in the
conversion of E3(ox).FAD complex to
E3(red).FAD intermediate
E3(red).FAD
Lipoamide-E2
PDC Catalysis: (5) E3.NAD+
E3(red).FAD
E3(ox).FADH2
E3(ox).FAD
- FAD oxidizes the thiol groups of reduced E3
dihydrolipoamide dehydrogenase [E3(red).FAD]
intermediate resulting in the formation of
E3(ox).FADH2 intermediate
- FADH2 within the E3(ox).FADH2 intermediate is
subsequently oxidized back to FAD by funneling its
electrons to NAD+ in an electron exchange reaction so
that NADH can be regenerated via the ETC (see §3.6)
- This yields active E3(ox).FAD complex with concomitant
release of NADH
- Recall that FAD is a more powerful oxidizing agent than
NAD+ (εFAD > εNAD+)
NAD+
Exercise 3.5a
- Write an equation for the pyruvate dehydrogenase complex (PDC)
reaction
- Describe the five reactions of the pyruvate dehydrogenase
complex (PDC)
- What cofactors are required? Which of these are prosthetic
groups?
Section 3.5b:
Krebs Cycle Reactions
Synopsis 3.5b
- Krebs cycle involves the oxidation of acetyl group of acetyl-coenzyme A (acetyl-CoA)
to CO2 with concomitant release of NADH, FADH2, and GTP
- Such oxidation of acetyl groups occurs via a “cycle” rather than a “pathway”—since both the
substrate and the product are identical (oxaloacetate), or simply put, the substrate
ultimately cycles to itself in a series of reactions—this is in contrast to a pathway in which a
substrate undergoes conversion to a chemically-distinct product!
- Krebs cycle is comprised of a total of eight enzymatic steps—excluding Step 0
for the synthesis of acetyl-CoA—and occurs within the mitochondrial matrix
- Overall reaction scheme is:
Acetyl-CoA + 3NAD+ + FAD + GDP + Pi <=> CoA + 3NADH + FADH2 + GTP + 2CO2
- In some bacteria, Krebs cycle runs counterclockwise in order to generate C compounds such
as acetyl-CoA solely from CO2 and H2O—in this so-called “reverse Krebs cycle”, a subset of
alternative enzymes are employed (like pathways, cycles are not wholly reversible!)
- In addition to Krebs cycle, other notable metabolic cycles include:
Cori cycle:
lactate  glucose
Cahill cycle:
alanine  glucose
Calvin cycle:
CO2  C compounds (C fixation—plants only!)
Krebs Cycle: Overview
- Widely considered as the “metabolic hub” due to
the fact that a major portion of macronutrients—
such as carbs, fats, and proteins—are oxidized via
Krebs cycle in order to generate free energy and
other metabolites needed to sustain life
- Named after Krebs, it is also known as the citric
acid cycle and tricarboxylic acid cycle
Hans Krebs
(1900-1981)
GDP
Krebs Cycle: Mono- and Dicarboxylic Acids
-
-
-
1:1
Formate
(Methanoate)
2:1
Acetate
(Ethanoate)
3:1
Propionate
(Propanoate)
-
4:1
Butyrate
(Butanoate)
-
-
2:2
Oxalate
(Ethanedioate)
-
-
-
-
3:2
Malonate
(Propanedioate)
5:1
Valerate
(Pentanoate)
4:2
Succinate
(Butanedioate)
-
5:2
Glutarate
(Pentanedioate)
α-Ketopropionate
Krebs Cycle:
A Logical Approach
α-Ketosuccinate
(Oxaloacetate)
L-α-Hydroxysuccinate
(L-Malate)
β-Hydroxy-β-carboxyglutarate
(Citrate)
D-α-Hydroxy-β-carboxyglutarate
(D-Isocitrate)
Didehydrosuccinate
(Fumarate)
α-Ketoglutarate
Succinate
Succinyl-CoA
Krebs Cycle Reactions: (1) Citrate Synthase
Catalyzes the condensation of acetyl-CoA and oxaloacetate (α-ketosuccinate) to produce citrate
(β-hydroxy-β-carboxyglutarate) and CoA:
(a) Acid-base catalysis mediated by active site D375/H274 residues produces a highly
reactive acetyl-CoA enolate nucleophile from acetyl-CoA and oxaloacetate
(b) Subsequent nucleophilic attack of acetyl-CoA enolate on the carbonyl group of
oxaloacetate generates citryl-CoA intermediate
(c) The citryl-CoA intermediate spontaneously hydrolyzes into citrate and free CoA
Krebs Cycle Reactions: (2) Aconitase
Catalyzes the isomerization of citrate (β-hydroxy-β-carboxyglutarate) to
isocitrate (α-hydroxy-β-carboxyglutarate):
(a) Dehydration of citrate resulting in the elimination of a H2O molecule
to generate cis-aconitate intermediate with a C=C double bond
(b) Hydration of the C=C double bond in cis-aconitate intermediate
generates isocitrate at the expense of a H2O molecule
Krebs Cycle Reactions: (3) Isocitrate Dehydrogenase
Catalyzes the oxidative decarboxylation of isocitrate (α-hydroxy-β-carboxyglutarate)
to α-ketoglutarate :
(a) Oxidation of isocitrate (using NAD+ as oxidizing agent) to oxalosuccinate
intermediate harboring a newly formed carbonyl group with concomitant
release of NADH
(b) Hyperpolarization of newly formed carbonyl group in oxalosuccinate by
Mn2+ ion facilitates the release of a CO2 molecule resulting in the formation
of a transient enolate intermediate—subsequent protonation of which
generates α-ketoglutarate
Krebs Cycle Reactions: (4) α-Ketoglutarate Dehydrogenase
Catalyzes the oxidative decarboxylation of α-ketoglutarate to succinyl-CoA:
(a) Oxidation of α-ketoglutarate (using NAD+ as oxidizing agent) facilitates the
release of a CO2 molecule
(b) Transfer of the thiol group of CoA-SH generates “high-energy” succinyl-CoA—
recall that thioester bonds (like phosphoanhydride bonds) are high-energy (their
hydrolysis releases lots of free energy to drive endergonic reactions)!
Krebs Cycle Reactions: (5) Succinyl-CoA Synthetase
Catalyzes the cleavage of “high-energy” succinyl-CoA to succinate coupled with the synthesis of
“high-energy” GTP via three steps:
(1) Condensation of Pi and succinyl-CoA to generate succinyl-phosphate (SucP) and CoA
(2) Transfer of phosphoryl group of SucP to a histidine on the enzyme so as to release succinate
(3) Transfer of phosphoryl group from the histidine on the enzyme to GDP to generate GTP
Krebs Cycle Reactions: (6) Succinate Dehydrogenase
Catalyzes dehydrogenation of succinate to fumarate (didehydrosuccinate)
using FAD (covalently bound to the enzyme via a histidine residue) as an
oxidizing agent (more powerful than NAD+):
(a) Electrons from succinate are funneled to FAD resulting in the
formation of a C=C double bond in fumarate
(b) Enzyme-bound FAD is reduced to FADH2
Krebs Cycle Reactions: (7) Fumarase
Catalyzes hydration of C=C double bond in fumarate (didehydrosuccinate)
to malate (α-hydroxysuccinate):
(a) Nucleophilic attack of an hydroxyl anion (from H2O) on C=C double
bond in fumarate generates a carbanion intermediate
(b) Protonation of carbanion intermediate generates malate
Krebs Cycle Reactions: (8) Malate Dehydrogenase
Catalyzes oxidation of malate (α-hydroxysuccinate) to
oxaloacetate (α-ketosuccinate):
(a) Electrons in the form of an hydride ion are funneled to
NAD+ so as to oxidize the hydroxyl moiety in malate to a
keto group in oxaloacetate
(b) NAD+ is reduced to NADH
Exercise 3.5b
- Explain why the citric acid cycle is considered to be the hub of
cellular metabolism
- What are the substrates and products of the net reaction
corresponding to one turn of the citric acid cycle?
- Draw the structures of the eight intermediates of the citric acid
cycle and name the enzymes that catalyze their interconversions
- Which steps of the citric acid cycle release CO2 as a product?
- Which steps produce NADH or FADH2? Which step produces GTP?
Section 3.5c:
Krebs cycle Regulation
Synopsis 3.5c
- Given that Krebs cycle plays a central role in the oxidation of
macronutrients to generate energy, it is imperative that it be
tightly regulated depending on cellular demands
- Key enzymes involved in Krebs cycle regulation are:
(a) Pyruvate dehydrogenase
(b) Citrate synthase
(c) Isocitrate dehydrogenase
(d) α-Ketoglutarate dehydrogenase
- Regulatory mechanisms include:
- Feedback inhibition
- Feedforth activation
- Allosteric regulation
- Post-translational modification (PTM)
-
Krebs cycle
Oxidation of acetyl group of Products
acetyl-CoA results in the liberation
of free energy and electrons
- Free energy is conserved in the
form of GTP—which can be
readily converted to ATP via the
action of NDK (nucleoside
diphosphate kinase):
ADP + GTP < = > ATP + GDP
- Electrons stored in the form of
NADH and FADH2 will be
funneled into the electron
transport chain (ETC) to reduce
O2 to H2O to generate ATP via
oxidative phosphorylation (§3.6)
Krebs cycle Thermodynamics (in cardiomyocytes)
Step
Enzyme
∆G° / kJ.mol-1
∆G / kJ.mol-1
0
Pyruvate dehydrogenase
<< 0
<< 0
1
Citrate synthase
-32
<< 0
2
Aconitase
+5
0
3
Isocitrate dehydrogenase
-21
<< 0
4
α-Ketoglutarate dehydrogenase
-33
<< 0
5
Succinyl-CoA synthetase
-2
0
6
Succinate dehydrogenase
+6
0
7
Fumarase
-3
0
8
Malate dehydrogenase
+30
0
- Recall that ∆G = ∆G° + RT lnKeq (§1.1)—where ∆G is the actual free energy change under
non-equilibrium (steady-state) conditions, and ∆G° is the standard free energy change @
equilibrium!
- Since living cells operate under steady-state rather than equilibrium setting, the free
energy changes associated with various Krebs cycle steps are largely concerned with ∆G
- Of the nine steps of Krebs cycle, only four (Steps 0, 1, 3 and 4) operate far from
equilibrium (∆G << 0)—implying that they are primarily responsible for flux control—ie
they are the rate-determining steps of Krebs cycle!
Krebs cycle Regulation: Key
Enzymes
0
a
Key enzymes involved in Krebs cycle regulation
are:
(a) Pyruvate dehydrogenase (Step 0)
(b) Citrate synthase (Step 1)
(c) Isocitrate dehydrogenase (Step 3)
(d) α-Ketoglutarate dehydrogenase (Step 4)
1
b
c 3
Activator
Inhibitor
Point of Inhibition
d
4
Krebs cycle Regulation: (a) Pyruvate Dehydrogenase
Acetyl-CoA | NADH
E1-OH (active)
Pyruvate | NAD+
PDK
PDP
E1-OPO32- (inactive)
Insulin | Ca2+
- Pyruvate dehydrogenase (E1) is a component of pyruvate dehydrogenase
complex (PDC) multi-enzyme system, which catalyzes the overall reaction:
Pyruvate + CoA + NAD+ => Acetyl-CoA + CO2 + NADH
- E1 is under tight regulation via three major mechanisms:
(1) Feedback Inhibition—PDC reaction products acetyl-CoA and NADH respectively compete with
corresponding substrates CoA and NAD+ for the E1 active site, thereby slowing down the enzyme as
the products accumulate
(2) Post-Translational Modification (PTM)—
- PDC reaction products acetyl-CoA and NADH activate pyruvate dehydrogenase kinase (PDK)—
which in turn phosphorylates a serine residue in E1 resulting in its inactivation
- Mitogenic signals (demanding energy production) such as insulin and Ca2+ reverse this
inactivation by virtue of their ability to activate pyruvate dehydrogenase phosphatase (PDP)—
which in turn dephosphorylates E1, thereby promoting its activation
(3) Feedforth Activation—Accumulation of pyruvate and NAD+ substrates serve as a signal for the
inhibition of PDK, thereby favoring the activation of E1 and driving the PDC reaction forward in the
direction of acetyl-CoA synthesis
Krebs cycle Regulation: (b) Citrate Synthase
Citrate | Succinyl-CoA | NADH
Oxaloacetate + Acetyl-CoA
Citrate
synthase
Citrate + CoA
Citrate synthase is regulated by:
Feedback Inhibition—inhibited
directly by its own product
citrate as well as downstream
products succinyl-CoA and NADH
Krebs cycle Regulation: (c) Isocitrate Dehydrogenase
ADP |
Ca2+
Isocitrate + NAD+
NADH | ATP
Isocitrate
dehydrogenase
Isocitrate dehydrogenase is regulated by:
(1) Feedback Inhibition—inhibited directly by
its own product NADH, which competes
with NAD+ for binding to the active site
(2) Allosteric Modulation—while ATP
allosterically inhibits the enzyme, ADP
exerts exactly the opposite effect—binding
of Ca2+ also augments its catalytic activity
α-Ketoglutarate + CO2 + NADH
Krebs cycle Regulation: (d) α-Ketoglutarate Dehydrogenase
Ca2+
α-Ketoglutarate + CoA + NAD+
Succinyl-CoA | NADH
α-Ketoglutarate
dehydrogenase
α-Ketoglutarate dehydrogenase is regulated by:
(1) Feedback Inhibition—inhibited directly by its
own products succinyl-CoA and NADH, which
respectively compete with α-ketoglutarate and
NAD+ substrates for binding to the active site
(2) Allosteric Modulation—Ca2+ binds and activates
the enzyme though the extent to which this
represents an allosteric effect is debatable
Succinyl-CoA + CO2 + NADH
Exercise 3.5c
- How much ATP can be generated from glucose when the citric acid
cycle is operating?
- Which steps of the citric acid cycle regulate flux through the cycle?
- Describe the role of ADP, Ca2+, acetyl-CoA, and NADH in regulating
pyruvate dehydrogenase and the citric acid cycle.