Part III => METABOLISM and ENERGY

§3.6 Krebs Cycle

§3.6a Pyruvate Oxidation

§3.6b Krebs Cycle Reactions

§3.6c 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) §3.5

- 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 mitochondrial matrix by the pyruvate-proton symporter located within the highly selective inner mitochondrial membrane (IMM)—the relatively-porous outer mitochondrial membrane (OMM) largely harbors 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 3.6a:

Pyruvate Oxidation

Synopsis 3.6a

- 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 acetyl 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

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 CoAresulting in the reduction of lipoamide to dihydrolipoamide

(3) Dihydrolipoamide/dihydrolipoyl dehydrogenase (E3)—oxidizes dihydrolipoamideto lipoamide in E2 using FAD and NAD+ as oxidizing agents

Lipoic Acid

Dihydrolipoamide

Reduction

Oxidation

Lipoic acid is covalently attached via an

amide linkage to a lysine residue in E2

dihydrolipoamide transacetylase—hence

often termed “lipoamide”

Lipoamide

LPA

PDC Catalysis: Catalytic Steps

1

2

3

4

5

Cys-Cys

disulfide bridge

Lipoamide (LPA)

E1 prosthetic group

(noncovalently bound)

E2 prosthetic group

(covalently bound)

E2 cosubstrate

E3 prosthetic group

(noncovalently bound)

E3 cosubstrate

PDC Catalysis: (1) E1.TPP

- 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)

Thiamine Pyrophosphate (TPP)

TPP consists of a central thiazole ring

harboring a carbanion flanked between

a pyrimidine ring and pyrophosphate

Pyrophosphate

PyrimidineThiazole

Hydroxyethyl-TPP.E1

PDC Catalysis: (2) E2-LPA

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-E2intermediate and regeneration of active TPP.E1

Lipoamide consists of lipoic acid

covalently linked via an amide

bond to the -amino group of a

lysine residue in E2

Lipoamide (LPA)

PDC Catalysis: (3) E2.CoA

- E2 dihydrolipoamide transacetylasecatalyzes 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

Coenzyme A (CoA)

Reactive thiol group

PDC Catalysis: (4) E3.FAD

- 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(ox).FAD E3(red).FADDihydrolipoamide-E2 Lipoamide-E2

Cys-Cys

disulfide bridge

PDC Catalysis: (5) E3.NAD+

- FAD oxidizes the thiol groups of reduced E3 dihydrolipoamidedehydrogenase [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, thereby yielding an active E3(ox).FAD complex with concomitant release of NADH

- Recall that FAD is a more powerful oxidizing agent than NAD+

(FAD > NAD+)—thus, it is energetically unfavorable for electrons to flow from FADH2 to NAD+—however, E3 overrides such energetic penalty by bringing NAD+ and FAD within close proximity to each other so as to allow the latter to serve as an electron conduit rather than an electron sink

E3(red).FAD E3(ox).FADE3(ox).FADH2

NAD+

Exercise 3.6a

- 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 3.6b:

Krebs Cycle Reactions

Synopsis 3.6b

- 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:Urea cycle: NH3 ureaCori cycle: lactate glucoseCahill cycle: alanine glucoseCalvin cycle: CO2 C compounds (C fixation—plants only!)

GDP

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)

Mono- and Dicarboxylic Acids

1:1

Formate

(Methanoate)

-

2:1

Acetate

(Ethanoate)

-

3:1

Propionate

(Propanoate)

-

4:1

Butyrate

(Butanoate)

-

5:1

Valerate

(Pentanoate)

-

2:2

Oxalate

(Ethanedioate)

--

3:2

Malonate

(Propanedioate)

- -

4:2

Succinate

(Butanedioate)

--

5:2

Glutarate

(Pentanedioate)

- -

Monocarboxylate Anions

Dicarboxylate Dianions

Succinate

Didehydrosuccinate

(Fumarate)

L--Hydroxysuccinate

(L-Malate)

-Ketosuccinate

(Oxaloacetate)-Hydroxy--carboxyglutarate

(Citrate)

D--Hydroxy--carboxyglutarate

(D-Isocitrate)

-Ketoglutarate

Succinyl-CoA

Krebs Cycle:

A Logical Approach

-Ketopropionate

Krebs Cycle Reactions: (1) Citrate Synthase

Catalyzes the condensation of acetyl-CoA and oxaloacetate (-ketosuccinate) to produce citrate (-hydroxy--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 (-hydroxy--carboxyglutarate) to isocitrate (-hydroxy--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

Is aconitase a mutase (since it overall mediates intramolecular transfer of a group)? But, aconitase also mediates bond cleavage to generate cis-aconitate intermediate? Mechanistically, aconitase is classified as a lyase rather than a mutase!

Catalyzes the oxidative decarboxylation of isocitrate (-hydroxy--carboxyglutarate) to -ketoglutarate :

(a) Oxidation of isocitrate (using NAD+ as oxidizing agent) to oxalosuccinateintermediate 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 -ketoglutarate

Krebs Cycle Reactions: (3) Isocitrate Dehydrogenase

Krebs Cycle Reactions: (4) -Ketoglutarate Dehydrogenase

Catalyzes the oxidative decarboxylation of -ketoglutarate to succinyl-CoA:

(a) Oxidation of -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 (-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

Fumarase belongs to the hydratase family of enzymes—addition of H2O to unsaturated bonds such as C=C

Krebs Cycle Reactions: (8) Malate Dehydrogenase

Catalyzes oxidation of malate (-hydroxysuccinate) to oxaloacetate (-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 3.6b

- 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 3.6c:

Krebs cycle Regulation

Synopsis 3.6c

- 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) -Ketoglutarate dehydrogenase

- Regulatory mechanisms include:- Feedback inhibition- Feedforth activation- Allosteric regulation- Post-translational modification (PTM)

Krebs cycle

Products

- Oxidation of acetyl group of 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 are stored in the form of

NADH and FADH2—which will be

ultimately funneled into the electron

transport chain (ETC) to reduce O2

to H2O to generate ATP via

oxidative phosphorylation (§3.7)

Krebs cycle Thermodynamics (in cardiomyocytes)

- 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—iethey are the rate-determining steps of Krebs cycle!

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 -Ketoglutarate dehydrogenase -33 << 0

5 Succinyl-CoA synthetase -2 0

6 Succinate dehydrogenase +6 0

7 Fumarase -3 0

8 Malate dehydrogenase +30 0

0

1

3

4

a

b

c

d

Krebs cycle Regulation: Key

Enzymes

Key enzymes involved in Krebs cycle regulation are:

(a) Pyruvate dehydrogenase (Step 0)(b) Citrate synthase (Step 1)(c) Isocitrate dehydrogenase (Step 3)(d) -Ketoglutarate dehydrogenase (Step 4)

Activator

Inhibitor

Point of Inhibition

Krebs cycle Regulation: (a) Pyruvate Dehydrogenase

- 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

E1-OH (active) E1-OPO32- (inactive)

PDK

PDP

Acetyl-CoA | NADHPyruvate | NAD+

Insulin | Ca2+

Krebs cycle Regulation: (b) Citrate Synthase

Oxaloacetate + Acetyl-CoA Citrate + CoA

Citrate | Succinyl-CoA | NADH

Citrate

synthase

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

Isocitrate + NAD+ -Ketoglutarate + CO2 + NADH

NADH | ATP

Isocitrate

dehydrogenase

ADP | Ca2+

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 ATPallosterically inhibits the enzyme, ADPexerts exactly the opposite effect—binding of Ca2+ also augments its catalytic activity

Krebs cycle Regulation: (d) -Ketoglutarate Dehydrogenase

-Ketoglutarate + CoA + NAD+ Succinyl-CoA + CO2 + NADH

Succinyl-CoA | NADHCa2+

-Ketoglutarate

dehydrogenase

-Ketoglutarate dehydrogenase is regulated by:

(1) Feedback Inhibition—inhibited directly by its own products succinyl-CoA and NADH, which respectively compete with -ketoglutarate andNAD+ 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

Exercise 3.6c

- 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.