TCA Cycle

Stage 1 of Cellular Respiration

oxidation of fuels to acetyl-CoA

generates ATP, NADH, FADH2

Stage 2 of Cellular Respiration

oxidation of acetyl groups to CO2 in the citric acid cycle (tricarboxylic acid (TCA) cycle, Krebs cycle)

nearly universal pathway

generates NADH, FADH2, and one GTP

Stage 3 of Cellular Respiration

electron transfer chain and oxidative phosphorylation

generates the vast majority of ATP from catabolism

Coenzyme A (CoA-SH)

coenzyme A has a reactive thiol (–SH) group that is critical to its role as an acyl carrier

– the –SH group forms a thioester with acetate in acetyl-CoA

Pyruvate is converted into ____ by process of _______

(acetyl-coA) (oxidation)

pyruvate dehydrogenase (PDH) complex = highly ordered cluster of enzymes and cofactors that oxidizes pyruvate in the mitochondrial matrix to acetyl-CoA and CO2

– the series of chemical intermediates remain bound to the enzyme subunits

– regulation results in precisely regulated flux

PDH Complex (function, reactants, and products)

Catalyzes an Oxidative Decarboxylation

An irreversible oxidation process in which the carboxyl group is removed, forming CO2

Procuces CO2, NADH,

PDH Complex Employs Three Enzymes

and Five Coenzymes to Oxidize Pyruvate

three enzymes:

– pyruvate dehydrogenase, E1

– dihydrolipoyl transacetylase, E2

– dihydrolipoyl dehydrogenase, E3

• five coenzymes:

– thiamine pyrophosphate (TPP)

– lipoate

– coenzyme A (CoA, CoA-SH)

– flavin adenine dinucleotide (FAD)

– nicotinamide adenine dinucleotide

PDH Complex Channels its Intermediates through ______ (number) Reactions

Five

Chemical Logic of the Citric Acid Cycle

• each step of the cycle involves either:

– an energy-conserving oxidation

– placing functional groups in position to facilitate

oxidation or oxidative decarboxylation

The Citric Acid Cycle Has _____ Steps

Eight

Net products of TCA cycle

3 NADH, 1 FADH₂, 1 GTP (or ATP), and 2 CO₂

STEP 1 Formation of Citrate

citrate synthase catalyzes the condensation of acetyl-CoA with oxaloacetate to form citrate

– large, negative ∆G′° is needed because [oxaloacetate] is very low

STEP 2: Formation of Isocitrate

aconitase (aconitate hydratase) = catalyzes the reversible transformation of citrate to isocitrate through the intermediate cis- aconitate

STEP 3: Oxidation of Isocitrate

isocitrate dehydrogenase catalyzes the oxidative

decarboxylation of isocitrate to α-ketoglutarate

– Mn2+ interacts with carbonyl group of the

oxalosuccinate and stabilizes transient enol

– specific isozymes for NADP+ (cytosolic and

mitochondrial) or NAD+ (mitochondrial)

STEP 4: Oxidation of α-Ketoglutarate

α-ketoglutarate dehydrogenase complex catalyzes the

oxidative decarboxylation of α-ketoglutarate to succinyl-CoA and CO2

– energy of oxidation is conserved in the thioester bond of succinyl-CoA

STEP 5: Conversion of Succinyl-CoA

succinyl-CoA synthetase (succinic thiokinase)

catalyzes the breakage of the thioester bond of succinyl-

CoA to form succinate

– energy released drives the synthesis of a

phosphoanhydride bond in either GTP or ATP (substrate

level phosphorylation)

STEP 6: Oxidation of Succinate

succinate dehydrogenase flavoprotein that catalyzes the

reversible oxidation of succinate to fumarate

– integral protein of the mitochondrial inner membrane in

eukaryotes

– contains three iron-sulfur clusters and covalently bound

FAD

Step 7: Hydration of Fumarate

fumarase = catalyzes the reversible hydration of fumarate to L-malate

transition state is a carbanion

Step 8: Oxidation of Malate

L-malate dehydrogenase = catalyzes the oxidation of L-malate to oxaloacetate, coupled to the reduction of NAD+

low [oxaloacetate] pulls the reaction forward

regenerates oxaloacetate for citrate synthesis

how much atp produced by nadh and fadh2

each NADH drives formation of ~2.5 ATP

each FADH2 drives formation of ~1.5 ATP

Citric Acid Cycle is (both)

amphibolic

anaplerotic reactions

chemical reactions that replenish intermediates

pyruvate carboxylase

catalyzes the reversible carboxylation of pyruvate by HCO3− to form oxaloacetate

most important anaplerotic reaction in mammalian liver, kidney, and brown adipose tissue

requires energy from ATP

allosterically activated by acetyl-CoA

Citric Acid Cycle Regulation points

PDH complex

citrate synthase

isocitrate dehydrogenase complex

α-ketoglutarate dehydrogenase complex

PDH complex activity is turned off when

ample fatty acids and acetyl-CoA are available as fuel

[ATP]/[ADP] and [NADH]/[NAD+] ratios are high

PDH complex activity is turned on when

energy demands are high

the cell requires greater flux of acetyl-CoA into the citric acid cycle

PDH kinase

inhibits the PDH complex by phosphorylation

allosterically activated by products of the complex

inhibited by substrates of the complex

PDH phosphatase

reverses the inhibition by PDH kinase

Citric Acid Cycle regulation occurs at strongly exergonic steps catalyzed by

citrate synthase

isocitrate dehydrogenase complex

α-ketoglutarate dehydrogenase complex

fluxes are affected by the concentrations of substrates and products

end products ATP and NADH are inhibitory

NAD+ and ADP are stimulatory

long-chain fatty acids are inhibitory