Pyruvate Oxidation and the Citric Acid Cycle Lecture 30 Pyruvate Oxidation and the Citric Acid Cycle Title slide

Aerobic Fate of Pyruvate: Overview Aerobic fate of acetylCoA - citric acid cycle

Mitochondria: Site of Oxidations Both the citric acid cycle enzymes and the pyruvate dehydrogenase complex are found in the mitochondrial matrix.

Pyruvate Oxidation to Acetyl-CoA Pyruvate dehydrogenase complexes uses 5 cofactors.

Pyruvate Dehydrogenase Complex Complex enzyme 60 polypeptides of 3 kinds In mitocondrion matrix Regulated Inhibited by NADH and GTP Stimulated by insulin Properties

Pyruvate Dehydrogenase Complex Three enzymes and 5 coenzymes The three components of the complex. I use E1, E2, & E3. This is the eukaryotic complex since there are regulatory proteins. Total MW: 4.5 x 106! Also present: regulatory protein kinase, phosphatase

Lipoic Acid: Acyl- and Redox Carrier A swinging arm of some 14 angstroms, undergoes redox, carries acyls. Note that the acyl is on the S from C-8.

Thiamine Pyrophosphate (TPP) TPP functions in the decarboxylation reaction. It loose a proton to yield a carbanion.

Pyruvate Dehydrogenase Mechanism The carbanion attacks the alpha C and forms an adduct.

Pyruvate Dehydrogenase Mechanism The carboxyl group is labilized and lost as carbon dioxide.

Pyruvate Dehydrogenase Mechanism Stabilized by resonance (shift between the two forms). Shifty objects are harder to pin down.

Pyruvate Dehydrogenase Mechanism Hydroxyethyl-TPP is an isolateable intermediate.

Pyruvate Dehydrogenase Mechanism Now they put the acyl group on the wrong S.

Pyruvate Dehydrogenase Mechanism The 8-acetyl lipoamide transfers the acetyl group to coenzyme A giving acetylCoA and producing dihydrolipoic acid.

Pyruvate Dehydrogenase Mechanism E3 dihydrolipoamide dehydrogenase reoxidizes the lipoamide.

Pyruvate Dehydrogenase Mechanism The special redox potential of this bound flavin allows it to transfer hydrogens to NAD+.

PDH: The Overall Reaction Acetyl-CoA enter Krebs cycle NADH passes e– to O2

Krebs Cycle Summary The Krebs citric acid acid or tricarboxylic acid cycle - three names.

Importance of the Krebs Cycle 1. Central energy-yielding path 2. Point of convergence of catabolism of fats, CHO, protein 3. Source of precursors for biosynthesis What it does.

Citrate Synthase: “Condensing Enzyme” ∆Gº= –32.2 kJ/mol Enzyme # 1 citrate synthase.

Aconitase ∆Gº = +13.3 kJ/mol Enzyme # 2 aconitase makes isocitrate. Functional Fe-S clusters. ∆Gº = +13.3 kJ/mol

Isocitrate Dehydrogenase Isocitrate dehydrogenase - an oxidative decarboxylation. The first carbon dioxide is lost. ∆Gº= –20.9 kJ/mol

-Ketoglutarate Dehydrogenase Complex a-oxoglutarate dehydrogenase - similar to the pyruvate dehydrogenase. Same cofactors. Another oxidative decarboxylation where the second carbon dioxide is lost. ∆Gº= –33.5 kJ/mol

-Ketoglutarate Dehydrogenase Complex Cofactors: FAD, NAD+, lipoate, TPP, CoASH Mechanism  PDH complex This enzyme complex does not involve a regulatory kinase and phosphatase.

Succinic Thiokinase ∆Gº= –2.9 kJ/mol A substrate level phosphorylation. ∆Gº= –2.9 kJ/mol

Succinic Thiokinase Multistep reaction: A phospho-histidine enzymeintermediate is formed.

Nucleoside Diphosphate Kinase ∆Gº= 0 Also uses CTP UTP So NTPs are in equilibrium with each other GTP and ATP are interconvertible.

Succinate Dehydrogenase Complex II of the electron transport chain is succinate dehydrogenase. ∆Gº= 0 Enzyme is membrane bound

Fumarase (Fumarate Hydratase) This is a stereospecific reaction. ∆Gº= –3.8 kJ/mol

Malate Dehydrogenase ∆Gº= +29.7 kJ/mol [OAA] normally < 10-6 M Now to close the circle and come back to the OAA starting compound. ∆Gº= +29.7 kJ/mol [OAA] normally < 10-6 M

Overall Reaction of Krebs Cycle The overall reactions.

Fate of NADH, FADH2ATP Each NADH3 ATP Each FADH22 ATP The reduced coenzymes are oxidized via the electron transport chain and coupled to oxidative phosphorylation. The lipoamide is oxidized by E3 which produces NADH. 2.5 & 1.5 ATPs instead of the 3 & 2 shown here. Each NADH3 ATP Each FADH22 ATP

Energy Yield from Glucose Oxidation The correct value is about 32.

Citric Acid Cycle: Metabolic Hub A metabolism center.

Regulation of the Citric Acid Cycle Factors that regulate the citric ac id cycle. Regulation of the Citric Acid Cycle

Krebs Cycle in Motion Jon Maber Dept of Biochemistry and Molecular Biology The University of Leeds, UK http://bmbwww.leeds.ac.uk/designs/tcasteps