pdh complex and tca cycle

PDH complex overview

pyruvate dehydrogenase complex exists in mitochondrial matrix

oxidative-decarboxylation of pyruvate (alpha-ketoacid) converts it to acetyl-CoA

5 coenzymes assisting PDH complex

thiamine pyrophosphate/TPP

lipoamide

NAD+

FAD

reduced Coenzyme A/CoASH

coenzyme

organic cofactor

where is TPP used?

pyruvate dehydrogenase/E1

where are lipoamide and CoASH used?

dihydrolipoamide transacetylase/E2

where are NAD+ and FAD used?

dihydrolipoamide dehydrogenase/E3

alpha-ketoacid dehydrogenase complex arrangement

three enzyme complexes: pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, alpha-ketoacid dehydrogenase → all oxidative decarboxylation reactions, utilize complex

E1: binds TPP, catalyzes decarboxylation

E2: transfers TPP bound ketoacid to thiol groups of lipoamide, adds CoA to ketoacid

E3: reduces FAD then reduces NAD+, using electrons obtained from reduction of lipoamide

thiamine pyrophosphate/TPP

stabilizes carbanion transition state

forms covalent adduct with pyruvate, provides e- delocalization

facilitates cleavage of C-C bonds by stabilizing carbanion intermediates

C2 on ring, between N and S, is deprotonated to form nucleophilic carbanion → attacks carbonyl carbon of pyruvate → ring stabilizes intermediate through resonance

tightly bound to E1

lipoamide

transfers acyl groups in alpha-keotacid dehydrogenases

has “swinging arm”: side chain in an E2 domain, interacts with active sites of E1/E3

accepts aldehyde fragment from TPP via oxidation of aldehyde and reduction of lipoamides disulfide group at the same time → generates acyl group which is transferred to CoA, pair of e- donated to lipoamide creates dihydrolipoamide

acts as e- carrier and an acyl group carrier

covalently bound to E2 by amide bond linking carboxyl of lipoid acid to lysine amino

coenzyme A/CoA

activates acyl groups (ex.: acetyl group from pyruvate)

acylation requires energy input: comes from oxidative decarboxylation of pyruvate

acylated = acyl-SCoA, unacylated = HS-CoA

acetyl-CoA is a thioester → high chemical potential due to resonance stabilization

• have C-SR instead of C-OR, larger size of S destabilizes bond = higher potential energy of acyl group transfer compared to an oxygen ester = more favorable deltaG of hydrolysis

• C-SR thioester bond is weaker than C-OR bond of esters, R-S- is good leaving group → acyl group is readily transferred

flavin adenine dinucleotide/FAD

derived from riboflavin/vitamin B

contains isoalloxazine ring system = 2 electron acceptor → ring attached to ribitol, ribitol attached to adenosine via pyrophosphate link

• riboflavin: ring attached to ribitol

• flavin mononucleotide: ring attached to ribitol attached to phosphate

undergoes two-electron oxidation and reduction reactions

tightly bound to E3: coenzyme cannot easily dissociate from enzyme → flavins cannot transfer electrons by moving from enzyme to enzyme

nicotinamide adenine dinucleotide/NAD+

electron carrier, participates in reversible two electron redox reactions via hydride transfers

• hydride transfer: both hydrogen and electron pair are transferred

substrate is oxidized → reduced NADH forms, leaves enzyme, is reoxidized by other systems

NAD+ and NADH redox

NAD+ and NADH redox

NAD+ and NADH redox

arsenic poisoning

arsenite inhibits all enzymes requiring lipoamide as a cofactor, thus E2 is affected and inhibited

arsenite forms complex with thiol/-SH groups of lipoamide and thus makes it unavailable as a cofactor (covalently modifies lipoamide groups)

pyruvate cannot be efficiently converted to acetyl-CoA, pyruvate accumulates, lactate accumulates, respiration cannot proceed

TCA overview

acetyl + 4 carbon oxaloacetate = 6 carbon citrate

rearranged into isocitrate

oxidative decarboxylation = e- transferred to NAD+ to form NADH/H+, CO2 released, ATP generated

succinate oxidized to oxaloacetate, FADH2 generated, NADH/H+ generated

citrate synthase

condenses activated acetyl group with oxaloacetate to form citrate

aconitase

rearranges OH on citrate to form isocitrate

requires Fe2+

isocitrate dehydrogenase

RATE LIMITING ENZYME

catalyzes oxidative decarboxylation of isocitrate: transfers electrons to NAD+ to generate NADH/H+, releases one carboxylate (COO-) as CO2

alpha-ketoglutarate dehydrogenase complex

structurally/functionally similar to PDH complex, requires same cofactors

causes second oxidative decarboxylation: removes one carboxylate (COO-) group as CO2, transfers electrons to NAD+ to generate NADH/H+, condenses CoASH to generate succinyl-CoA

succinyl-CoA synthetase

energy from succinyl-CoA thioester bond generates ATP from ADP + Pi.

substrate-level phosphorylation

succinate dehydrogenase

transfers pair of electrons from acetyl group to FAD to generate FADH2 and fumarate

resides in inner mitochondrial membrane, unlike all others in mitochondrial matrix

fumarase

incorporates H2O into fumarate to generate l-malate

l-malate dehydrogenase

transfers final electron pair from acetyl group to NAD+ to generate NADH/H+ and regenerate oxaloacetate

3 stages of respiration

1: generation of acetyl-CoA + two electrons

• mitochondrial matrix

2: oxidation of acetyl-CoA into 2 CO2 + 8 electrons, NADH + FADH2

• mitochondrial matrix

3: reoxidation of reduced e- carries provides energy for ATP synthesis, NAD+ + FAD

• mitochondrial inner membrane

oxidation and reduction

oxidation removes e- from substrate (donor) and gives to carrier (acceptor)

• metabolic oxidations = loss of hydrogen from substrate → catalyzed by dehydrogenases

reduction gives e- to substrate

dicarboxylic acids: citrate, succinate, fumarate, malate

act catalytically

citrate

succinate

fumarate

malate

acetate

malonate

TCA cycle regulation

corresponds to rate of electron-transport chain → regulated by ATP/ADP ratio and rate of ATP use

messengers: ADP, NADH

• increased ADP activates, increased NADH inhibits

isocitrate dehydrogenase = principle enzyme regulator → activated by increased ADP/Ca2+, inhibited by increased NADH

alpha-ketoglutarate dehydrogenase complex: activated by Ca2+, inhibited by NADH

malate dehydrogenase: inhibited by NADH

citrate synthase: inhibited by high citrate concentrations

TCA cycle intermediates

malate: produced in liver during fasting from glucogenic precursors, leaves mitochondria for cytosolic gluconeogenesis

liver uses other intermediates to synthesis carbon skeletons of AA’s

succinyl-CoA: may be removed to form heme in liver, bone marrow

alpha-ketoglutarate: converted to glutamate in brain, then to GABA

alpha-ketoglutarate: converted to glutamate in skeletal muscle, transported through body