W3 Tricarboxylic Acid Cycle

Question 1

What is the pyruvate dehydrogenase multienzyme complex (PDC)

Answer 1

used for conversion of pyruvate to acetyl coA by oxidative decarboxylation

a cluster of 3 enzymes: pyruvate dehydrogenase, dihydrolipoul transacetylase, dihydrolipoyl dehydrogenase

require 5 cofactors: thiamines pyrophosphate (TPP), lipoic acid, coenzyme A, FAD and NAD+

reaction occurs in mitochondrial matrix

Question 2

properties of acetyl coA

Answer 2

functional role: acetyl group carrier

reactive group: thiol (-SH)

acetyl group covalently linked to thiol > forming high energy thioester (hydrolysis is -31.5)

formation of thioester bond in metabolic intermediate conserves a portion of the free energy released during oxidation of a metabolic fuel

high free energy released upon hydrolysis of thioester bond

Question 3

Why is acyl group readily transferred to other metabolites

Answer 3

Most esters like oxygen esters gave two resonance forms > partial double bond character

lack of this resonance in thioesters in SR bond of acyl coA makes bond weaker > thiolalkoxide ion is a good leaving group in nucleophilic displacement reactions

Question 4

properties and function of thiamine pyrophosphate (TPP)

Answer 4

pyrophosphate of thiamine (vitamin B1)

function: transfer of an activated aldehyde unit

functional group: thiazolium ring,which has an acidic proton at C2 > loss of proton produces carbanion (the active species)

reaction catalyzed by pyruvate dehydrogenase

Question 5

how does TPP function in the pyruvate dehydrogenase reaction

Answer 5

1. nucleophilic attack by ylide form of TPP on carbonyl carbon of pyruvate > release CO2 and form hydroxyethyl TPP (decarboxylation)
2. Transfer of hydroxyethyl group to lipoamide moiety (hydroxyethyl carbonaion on lipoamide disulfide) > elimination of TPP to form acetyl thioester on dihydrolipoamide
3. transfer of acetyl group to coA > produce acetyl coA and dihydrolipoamide

Question 6

colour of oxidized and reduced forms of FAD

Answer 6

oxidized form (FAD): yellow with wavelength max = 450nm

reduced form (FADH2): colourless

Question 7

summary of pyruvate dehydrogenase complex reaction

Answer 7

1. pyruvate reacts with TPP > decarboxylation > hydroxyethyl-TPP of E1
2. aldehyde group is transferred to one lipoamide cofactor of E2 and simultaneously oxidized to acetyl
3. acetyl group is transferred to the next lipoamide cofactor > transferred to CoA-SH > form acetyl coA
4. E3 oxidizes lipoamide by transferring 2 H atoms to FAD
5. FADH2 oxidized by NAD+ > enzyme complex ready for next cycle

Question 8

results of TPP deficiency

Answer 8

vitamin B1 deficiency > TPP deficiency

pyruvate dehydrogenase (E1) inhibited > pyruvate oxidation inhibited > neurological and cardiovascular disorder

detection: increase in pyruvate in blood

Question 9

step 1 of TCA cycle

Answer 9

acetyl coA adds its two carbon group to oxaloacetate via citrate synthase > produce citrate

Question 10

step 2 of TCA cycle

Answer 10

citrate is tertiary alcohol > cannot be oxidised > citrate must be converted to isocitrate (chiral secondary alcohol)

reaction catalysed by aconitase

citrate > dehydration to form cis-aconitase intermediate > rehydration to form isocitrate

Question 11

step 3 of TCA cycle

Answer 11

NAD+ dependent isocitrate dehydrogenase catalyze oxidation of isocitrate to oxalsuccinate (produce NADH) > decarboxylation of carboxyl group (release CO2) > form alpha ketoglutarate

keto beta group to the carboxyl group facilitates the decarboxylation by acting as a electron sink

oxidation occurs with reduction of NAD+

Question 12

step 4 of TCA cycle

Answer 12

alpha ketoglutarate dehydrogenase catalyses oxidative decarboxylation of alpha ketoglutarate > release CO2 and NADH (mediated by PDC) > produce succinyl CoA

Question 13

step 5 of TCA cycle

Answer 13

succinyl coA synthase catalyses formation of succinyl phosphate > formation of phosphoryl-His intermediate > transfer of phosphoryl group to GDP to form GTP > GTP can exchange its terminal phosphoryl group with ADP via nucleoside diphophate kinase > produce ATP

succinate is formed

Question 14

step 6 of TCA cycle

Answer 14

succinate dehydrogenase oxidises succinate to fumarate > produce FADH2

enzyme has FAD covalently bound > can be reoxidised by coenzyme Q in ETC

enzyme is the only membrane bound TCA cycle enzyme

Question 15

step 7 of TCA cycle

Answer 15

hydration of fumarate to malate by fumarase

Question 16

step 8 of TCA cycle

Answer 16

regeneration of oxaloacetate from malate by malate dehydrogenase

1 NADH produced

Question 17

products of one TCA cycle

Answer 17

3 NADH, 2 CO2, 1 FADH2, 1 ATP

Question 18

net products from both glycolysis and TCA cycle

Answer 18

6 CO2, 10 NADH2, 2 FADH2, 4 ATP

Question 19

major regulatory interactions in TCA cycle

Answer 19

oxidation of acetyl coA can only go as fast as electrons from NADH enter ETC which is controlled by ATP and ADP content of cells

Increase NADH concentration > isocitrate dehydrogenase, alpha ketoglutarate dehydrogenase and malate dehydrogenase inhibited

during muscular contraction, increased Ca2+ concentration actibates isocitrate DH, alpha keto glutarate DH and pyruvate DH

Question 20

anaplerotic pathways to replenish TCA cycle intermediates

Answer 20

pyruvate carboxylase converts pyruvate to replenish oxaloacetate

glutamate reversibly converted to alpha ketoglutarate

some compounds enter TCA cycle at level of succinyl CoA

other amino acids also degraded to fumarate