35a Pyruvate Oxidation 35b Krebs Cycle Reactions 35c Krebs Cycle Regulation III METABOLIC BIOCHEMISTRY Sources of Acetyl Group of AcetylCoA Acetyl group of acetylCoA can be derived ID: 907962
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Slide1
§3.5 Krebs Cycle §3.5a Pyruvate Oxidation §3.5b Krebs Cycle Reactions §3.5c Krebs Cycle Regulation
III. METABOLIC BIOCHEMISTRY
Slide2Sources of Acetyl Group of Acetyl-CoA
Acetyl group of
acetyl-CoA
can be derived
from the breakdown of:(1) Carbs (glucose) §3.1(2) Lipids (fatty acids) §3.3(3) Proteins (amino acids) §3.4Under normal physiological conditions, the acetyl group of acetyl-CoA is largely derived from pyruvate (the end product of glycolysis)—see §3.1Pyruvate 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
Slide3§3.5a Pyruvate Oxidation
Slide4Synopsis 3.5a
A major source of acetyl group of acetyl-CoA
entering the Krebs cycle is derived from
pyruvate generated from carbohydrates via
glycolysisTransfer 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 PDCEach 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 cycleOverall reaction (irreversible) catalyzed by PDC is: Pyruvate + CoA + NAD+ ==> Acetyl-CoA + CO2 + NADH
Slide5PDC 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 + NADHThis 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+
Slide6PDC Catalysis: (Dihydro)lipoamide
Specific roles of the three
subenzymes
in PDC are:
Pyruvate dehydrogenase (E1)—oxidizes/decarboxylates pyruvate, transferring the acetyl group to bound TPP cofactor 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 dihydrolipoamideDihydrolipoamide/dihydrolipoyl dehydrogenase (E3)—oxidizes dihydrolipoamide to lipoamide in E2 using FAD and NAD+ as oxidizing agents Lipoic Acid
D
ihydrolipoamide
Reduction
Oxidation
Lipoic
acid is covalently attached via an amide linkage to a
lysine
residue in E2
dihydrolipoamide
transacetylase
—hence often termed “
lipoamide
”
Lipoamide
Slide7LPA
PDC Catalysis: Catalytic Steps
1
2
3
4
5
Cys-Cys
disulfide bridge
Lipoamide
(LPA)
E1 prosthetic group
(
n
oncovalently
bound)
E2 prosthetic group (covalently bound)
E2
c
osubstrate
E3 prosthetic group
(
n
oncovalently
bound)
E3
c
osubstrate
Slide8PDC Catalysis: (1) E1.TPP E1 pyruvate dehydrogenase
(which harbors TPP cofactor)
decarboxylates
pyruvate
resulting in the formation of ethylhydroxy-TPP.E1 carbanion intermediate The reaction is mediated by the nucleophilic attack of TPP carbanion on the carbonyl atom of the acetyl moiety on pyruvateRecall that TPP is also required for the decarboxylation of pyruvate to acetaldehyde by pyruvate decarboxylase during alcoholic fermentation (see §3.1c) Thiamine Pyrophosphate (TPP)TPP consists of a central thiazole ring harboring a carbanion flanked between a pyrimidine ring and pyrophosphatePyrophosphate
Pyrimidine
Thiazole
Ethylhydroxy
-
TPP.E1
Slide9Ethylhydroxy-TPP.E1
PDC Catalysis: (2) E2-LPA
Hydroxyethyl moiety of
ethylhydroxy-TPP.E1
intermediate is transferred to E2 dihydrolipoamide transacetylase harboring a lipoamide cofactor (lipoamide-E2) via two steps:Hydroxyethyl carbanion in the ethylhydroxy-TPP.E1 complex launches nucleophilic attack on the lipoamide disulfide in lipoamide-E2Hydroxyethyl 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
Lipoamide consists of lipoic acid covalently linked via an amide bond to the
-amino group of a lysine residue in E2
Lipoamide (LPA)
Slide10PDC Catalysis: (3) E2.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 Coenzyme A (CoA)Reactive thiol group
Slide11PDC 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 reactionIn 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).FAD
Dihydrolipoamide-E2
Lipoamide-E2
Cys-Cys
disulfide bridge
Slide12PDC Catalysis: (5) E3.NAD+
FAD oxidizes the
thiol
groups
of reduced E3 dihydrolipoamide dehydrogenase [E3(red).FAD] intermediate resulting in the formation of E3(ox).FADH2 intermediateFADH2 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 NADHRecall 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).FAD
E3(ox).FADH
2
NAD
+
Slide13Exercise 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?
Slide14§3.5b Krebs Cycle Reactions
Slide15Synopsis 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 GTPSuch 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 matrixOverall reaction scheme is: Acetyl-CoA + 3NAD+ + FAD + GDP + Pi <=> CoA + 3NADH + FADH2 + GTP + 2CO2In 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:
NH
3
urea
Cori cycle: lactate
glucose
Cahill cycle: alanine
glucose
Calvin cycle: CO
2
C compounds (C fixation—plants only!)
Slide16GDP
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 lifeNamed after Krebs, it is also known as the citric acid cycle and tricarboxylic acid cycle Hans Krebs(1900-1981)
Slide17Krebs Cycle Metabolites ᴙ Us!1:1Formate(Methanoate)
-
2:1
Acetate
(Ethanoate)-3:1Propionate(Propanoate)-4:1Butyrate(Butanoate)-5:1Valerate(Pentanoate)-2:2Oxalate(Ethanedioate)--3:2Malonate(Propanedioate)--4:2
Succinate
(
Butanedioate
)
-
-
5:2
Glutarate
(
Pentanedioate
)
-
-
Monocarboxylate
Anions
Dicarboxylate
Dianions
6:1
Caproate
(
Hexanoate)-
Slide18Succinate
Didehydrosuccinate
(
Fumarate
)L--Hydroxysuccinate(L-Malate)-Ketosuccinate(Oxaloacetate)-Hydroxy--carboxyglutarate(Citrate)D--Hydroxy--carboxyglutarate(D-Isocitrate)-KetoglutarateSuccinyl-CoAKrebs Cycle: A Logical Approach-Ketopropionate
GDP
Slide19Krebs Cycle
Reactions: (1) Citrate Synthase
Catalyzes the condensation of
acetyl-CoA
and oxaloacetate (-ketosuccinate) to produce citrate (-hydroxy--carboxyglutarate) and CoA:Acid-base catalysis mediated by active site D375/H274 residues produces a highly reactive acetyl-CoA enolate nucleophile from acetyl-CoA and oxaloacetate Subsequent nucleophilic attack of acetyl-CoA enolate on the carbonyl group of oxaloacetate generates citryl-CoA intermediateThe citryl-CoA intermediate spontaneously hydrolyzes into citrate and free CoA
Slide20Krebs Cycle Reactions: (2) Aconitase
Catalyzes the isomerization of
citrate (
-
hydroxy--carboxyglutarate) to isocitrate (-hydroxy--carboxyglutarate):Dehydration of citrate resulting in the elimination of a H2O molecule to generate cis-aconitate intermediate with a C=C double bondHydration 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!
Slide21Catalyzes the oxidative decarboxylation of isocitrate (-hydroxy--carboxyglutarate) to
-
ketoglutarate
:Oxidation of isocitrate (using NAD+ as oxidizing agent) to oxalosuccinate intermediate harboring a newly formed carbonyl group with concomitant release of NADHHyperpolarization 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
Slide22Krebs Cycle Reactions: (4) -Ketoglutarate Dehydrogenase
Catalyzes the oxidative decarboxylation of
-
ketoglutarate
to succinyl-CoA:Oxidation of -ketoglutarate (using NAD+ as oxidizing agent) facilitates the release of a CO2 moleculeTransfer 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)!
Slide23Krebs 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
Slide24Krebs 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+):Electrons from succinate are funneled to FAD resulting in the formation of a C=C double bond in fumarate Enzyme-bound FAD is reduced to FADH2
Slide25Krebs Cycle Reactions: (7) Fumarase
Catalyzes hydration of C=C double bond in
fumarate
(
didehydrosuccinate) to malate (-hydroxysuccinate): Nucleophilic attack of an hydroxyl anion (from H2O) on C=C double bond in fumarate generates a carbanion intermediate Protonation of carbanion intermediate generates malateFumarase belongs to the hydratase family of enzymes—addition of H2O to unsaturated bonds such as C=C
Slide26Krebs Cycle Reactions: (8) Malate Dehydrogenase
Catalyzes oxidation of
malate (
-
hydroxysuccinate) to oxaloacetate (-ketosuccinate):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 oxaloacetateNAD+ is reduced to NADH
Slide27Exercise 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
interconversionsWhich steps of the citric acid cycle release CO2 as a product?Which steps produce NADH or FADH2? Which step produces GTP?
Slide28§3.5c Krebs cycle Regulation
Slide29Synopsis 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) -Ketoglutarate dehydrogenaseRegulatory mechanisms include:Feedback inhibitionFeedforth activationAllosteric regulationPost-translational modification (PTM)
Slide30Krebs 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 + GDPElectrons 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 (§3.6)
Slide31Krebs Cycle Thermodynamics (in cardiomyocytes)
Recall that
G = G + RT
lnK
eq (§1.3)—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 GOf 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! StepEnzymeG / kJ.mol-1G / kJ.mol-10Pyruvate dehydrogenase << 0<< 01Citrate synthase-32<< 02Aconitase+503Isocitrate dehydrogenase-21<< 04
-
Ketoglutarate
dehydrogenase
-33
<< 0
5
Succinyl
-CoA
synthetase
-2
0
6
Succinate dehydrogenase
+6
0
7
Fumarase
-3
0
8Malate dehydrogenase+300
Slide320
1
3
4
a
b
c
d
Krebs Cycle
Regulation:
Key
Enzymes
E
nzymes involved in Krebs cycle regulation are
:
Pyruvate dehydrogenase (Step 0)
Citrate synthase (Step 1)
Isocitrate
dehydrogenase (Step 3)
-
Ketoglutarate
dehydrogenase (Step 4)
Activator
Inhibitor
Point of Inhibition
Slide33Krebs 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 + NADHE1 is under tight regulation via three major mechanisms: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 inactivationMitogenic signals (demanding energy production) such as insulin and
Ca
2+
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 | NADH
Pyruvate | NAD
+
Insulin | Ca
2+
Slide34Krebs Cycle Regulation: (b) Citrate Synthase
Oxaloacetate + Acetyl-CoA
Citrate + CoA
Citrate |
Succinyl-CoA | NADHCitrate synthaseCitrate synthase is regulated by:
Feedback Inhibition
—inhibited directly by its own product
citrate
as well as downstream
products
succinyl
-CoA
and
NADH
Slide35Krebs Cycle Regulation: (c) Isocitrate Dehydrogenase
Isocitrate + NAD
+
-Ketoglutarate
+ CO2 + NADHNADH | ATPIsocitrate dehydrogenase
ADP | Ca
2+
Isocitrate
dehydrogenase is regulated by:
Feedback Inhibition
—inhibited directly by its own product
NADH
, which competes with
NAD
+
for
binding to the
active
site
Allosteric Modulation
—while
ATP
allosterically
inhibits the enzyme,
ADP
exerts exactly the opposite effect—binding of
Ca
2+
also augments its catalytic activity
Slide36Krebs Cycle Regulation: (d) -Ketoglutarate Dehydrogenase
-Ketoglutarate
+ CoA + NAD
+
Succinyl-CoA + CO2 + NADHSuccinyl-CoA | NADHCa2+
-Ketoglutarate
dehydrogenase
-
Ketoglutarate
dehydrogenase is regulated by:
Feedback Inhibition
—inhibited directly by its own products
succinyl
-CoA
and
NADH
, which respectively compete with
-
ketoglutarate
and
NAD
+
substrates for binding to the
active
site
Allosteric Modulation
—
Ca
2+
binds and activates the enzyme though the extent to which this represents an allosteric effect is debatable
Slide37Exercise 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, Ca
2+, acetyl-CoA, and NADH in regulating pyruvate dehydrogenase and the citric acid cycle.