lec. 16 TCA CYCLE part 1

converting pyruvate into acetyl-coa via PDH

Tricarboxylic acid (TCA) cycle

• central pathway in aerobic metabolism

• catabolism of carbohydrates, fatty acids, and emino acids

• eight biochemical rxns/steps

• oxidize 2 carbon contained in acetyl-coa to 2 carbon dioxide molexules

  • 3 nadh, 1 fadh2, 1 atp via substrate-level phsophorylation

cellular respiration

cells oxidize biological fuels, transferring electrons to molecular oxygen, and captures released energy as ATP

three stages of cellular respiration

TCA cycle, electron transport chain, protons flow back into matrix (atp synthesis)

last two stages fall under oxidative phosphorylation

aerobic respiration much more energy from glucose

• 30-32 ATP per glucose in aeobic respiration

  • 2 ATP from glycolysis

  • 2 GTP (2 ATP equiv) from TCA cycle

  • around 26-28 ATP from oxidative phosphorylation

coenzyme A carries acyl groups

• business end of coa → thiol bond that can form thiolester bond with carboxylic acid

• synthesis of coa requires ribose 5-phosphate

thioester hydrolysis has a large negative standard free energy change (-31.4 kJ/mol)

• high energy bond → hydrolysis of product is stabilized relative to the substrate

why is thioester hydrolysis more favorable?

• thioesters are less resonance-stabilized than comparable oxygen esters

• hydrolysis produces same resonance-stabilized carboxylate

• therefore, thioester hydrolysis is more energetically favorable than oxygen ester hydrolysis

which biological fuels converge acetyl-coa

• carbohydrates

• fatty acids

• amino acids

acetyl-coa is a central metabolic branch point

• TCA cycle: acetyl-coa oxidized into CO2 → NADH + FADH2, and ATP via oxidative phosphorylation

• acetyl-coa used for biosynthetic reactions

• CANNOT be converted back to pyruvate → not a precursor for gluconeogenesis

pyruvate oxidation links glycolysis to the TCA cycle

• pyruvate (3C) → acteyl-coa (2C)

  • 1C lost as CO2

  • NADH extracted (2e extracted)

• large negative G = irreversible

• highly regulated in metabolic pathways

pyruvate oxidation and the TCA cycle occur in the mitochondrial matrix

• pyruvate-proton symporter: pyruvate that has been formed from glycolysis is transported to the mitochondrial matrix

structure of the mitochondria

• outer membrane

• intermembrane

• inner membrane

• mitochondrial matrix

outer membrane of the mitochondria

permeable to ions and small molecules

inter membrane of the mitochondria

resembles cytosol because porousness of outer membrane

high pH means low H

low pH means high H

inner membrane of the mitochondria

highly selective; relatively impermeable

has specific transport proteins

contains the electron transport chain and ATP synthesis

mitochondrial matrix of the mitochondria

enclosed within the inner mitochondria

low H+ concentration → high pH

site of pyruvate oxidation and most TCA cycle reactions

site of fatty acid beta-oxidation and some reactions of urea cycle

pyruvate dehydrogenase complex (PDH)

converts pyruvate into acteyl-coa

highly irreversible

contains five co-enzymes/co-factors:

• catalytic cofactors: thiamine, pyrophosphate, lipoamide, FAD

  • starts and ends the same; just different during the rxn(s)

• stoichiometric cofactors: CoA and NAD+

  • function as substrates; will be changed at the end

lipoamide (catalytic co-factor)

• flexible carrier of acyl groups and electrons during catalysis

• backbone of lipoic acid → fatty acid that 8-carbon

• 5-membered ring disulfide bond → can open up to be reduced into 2 or 3 thiol groups

flavin cofactors (FMN or FAD) can accept one or two electrons

• can accept 1 or 2 electrons

  • 2 electrons and 2 H → fully reduced

  • 1 electron and 1 H → semi-reduced, semiquinone FADH or FMNH

• ability to accept 1 or 2 e makes it a good intermedeiate between one electron acceptor or donor

difference between flavin cofactors and NAD

covalently attached or tightly bound to the enzyme; → cannot be easily dissociable

• once FMN or FAD is fully reduced, it cannot disassociate from the enzyme to pass its electrons downstream

  • reduced FADH2 must pass electrons to something equally soluble (ex. NAD)

    • oxidized again to do rxn

PDH complex composed of three enzymes

• E1 (pyruvate dehydrogenase component)

  • catalyzes oxidative decarboxylation of pyruvate

  • prosthetic group = TPP

• E2 (dihydrolipoyl transacetylase)

  • catalyzes transfer of acetyl group to CoA

  • prosthetic group = lipoamide

• E3 (dihydrolipoyl dehydrogenase)

  • regeneration of oxidized form of lipoamide

  • prosthetic group = FAD

    • oxidizes the reduced FADH2

thiamine pyrophosphate (TPP)

• binds to E1 (pyruvate dehydrogenase component)

• supports decarboxylation rxn of pyruvate

• carries 2-carbon fragment as hydryoxythyl-TPP

Lipoamide

• covalently attached to E2 (dihydrolipoyl transacetylase)

  • helps to transfer acyl group to CoA

• accepts and oxidizes hydroxyethyl group of TPP → acetyllipoamide

  • transfer of acetyl group to reduced form of lipoamide by E3

CoA

• cosubstrate of E2; accepts acetyl group from acetyl lipoamide → acetyl-coa

Flavin adenin nucleotide (FAD)

tightly bound to E3

reoxidizes reduced lipoamide to reduced FADH2 forming

nicotinamide adenine dinucleotide (NAD)

co-substrate of E3; reoxidzes FADH2 to FAD

E1: decarboxylation of pyruvate

• E1 catalyzes decarboxylation rxn by oxidation of 2-carbon fragment

• TPP to stabilize carbon ion formed during C-C bond cleavage

E2: oxidation of hydroxyethyl-TPP and transfer to lipoamide

• oxidant = lipoamide

  • oxidizes hydroxyethyl-TPP’s carbide to form carbanion

  • disulfide bond reduced → generates 2 thiol groups → acetyl transferred to one of the free thiol groups

  • forms acylipoamide → regenerating TPP

E2: transfer of acetyl group to CoA

• E2 transfers acetyle group from acetyllipoamide to CoA

• generates CO2 in the process

• regenerates disulfide bond form of lipoamide + reduced dihydrolipoamide

E3: regen of oxidized lipoamide

• FAD bound to E3 accepts electrons from reduced lipoamide

  • regenerating oxidized lipoamide → FADH2 transfers electrons to NAD → NADH and FAD regenerated

• NAD oxidizes dihydrolipoamide, forming NADH

PDH complex is organized as a large multi-enzyme complex

• E2 = central/core

• E1 and E3 = around periphery

• E2’s domain is flexible; can move among active sites of E1, E2, E3

flexible lipoamide arm moves between active sites

net reaction of PDH complex

pyruvate + CoA-SH + NAD → acetyl-coa + CO2 + NADH + H

^happens two times because glucose produces 2 pyruvate

therefore we get a total of 2 acetyl-coa and 2 nadh and 2 CO2 is released

allosteric regulation of PDH complex

ATP inhibts reaction while AMP promotes reaction because it is promoted by low energy in teh cell

also inhibited by its producs and fatty acids

activated by CoA and NAD (substrates)

regulation of PDH complex by protein phosphrylation

PDH complex can also be regulated by covalent post-translation modification

pyruvate dehydrogenase kinase (PDK)

• catalyzes phosphorylation of E1 enzyme

  • is reversible

when PDK phosphrylates E1

PDH-P is inactive

when PDK-1 dephosphrylates E1…

PDH is active

PDK is activated by

• ATP

• NADH

• acetyl-coa

PDK is inhibited by …

• pyruvate

• ADP

pyruvate dehydrogenase phosphotase is activated by

Ca2+ contracting in muscles because they need more energy

PDH deficiency

• less acetyl-coa because pyruvate cannot convert it

• less glucose-derived carbon entering the TCA cycle

• more pyruvate in cytosol makes over production of alanine

• less aerobic ATP production from glucose

what helps PDH deficiency

• thiamine overdose

• ketone bodies

warburg effect: aerobic glycolysis in cancer cells

• what is it: cancer cells take large amounts of glucose in the cell but experience decreased pyruvate oxidation → does not enter TCA cycle

• increased glucose uptake, glycolytic flux, and lactate fermention

• pyruvate turnes to lactate despite O2 being available