1. Ferchmin 2017
Index:
Pyruvate dehydrogenase complex, structure, cofactors, mechanism, regulation.
TCA or Kreb’s cycle, function and regulation.
Asymmetric breakdown of a symmetric citrate molecule.
“Accounting” of ATP synthesized, from glycolysis to respiratory chain.
Murphy’s Law and the missing 10 molecules of ATP per glucose.
How to avoid losing your carbons during the exam.

2. Steps shown and enzymes involved.
1 and 2) Pyruvate dehydrogenase (not decarboxylase).
3 and 4) Dihydrolipoyl transacetylase.
5) Dihydrolipoyl dehydrogenase
How come NAD++H
transfers H to FAD?
Fully reduced lipoic
The –SH reduces the other –S- of lipoic acid

3. The pyruvate dehydrogenase complex is s true multienzymatic complex
Model of the PDC of E. coli.
Red: E1 pyruvate dehydrogenase
Yellow: E2 transacetylase
Green: E3 dihydrolipoyl dehydrogenase
Electron micrography of
the PDC of E. coli.

4. Regulation of the PDC by allosteric and covalent modulation
The enzymes involved
Coenzymes
Description
Activators
Inhibitors
1) Pyruvate dehydrogenase (E1)
Thiamine pyrophosphate
(Vitamin B1)
Decarboxylation of pyruvate, E1α and E1β, phosphorylation site of PDC.
30 subunits
AMP
energy
GTP
2) Lipoate transacetylase (E2 )
Lipoic acid
Dehydrogenation of hydroxyethyl and transfer of acetyl to CoA.
60 subunits
CoA
TCA substrate
CoA~Acetyl
3) Dihydrolipoyl dehydrogenase (E3)
NAD+ and FAD
(Niacin & B2)
Regeneration of the lipoate and reduction of NAD+ to NADH.
10 subunits
NAD+
Reducing power
NADH;
The regulatory subunits of the PDC are intrinsic to the complex
1) Pyruvate dehydrogenase kinase inactivates the PDC Complex by phosphorylation of three serines on E1α.
PRODUCTS
ATP Acetyl CoA NADH
SUBSTRATES
ADP; pyruvate;
CoA; NAD+
2) Pyruvate dehydrogenase phosphatase removes the inhibition imposed by the phosphorylation through hydrolysis of the Pi.
Ca2+
insulin
NADH
Ca2+ in muscle & insulin in liver
Insulin (means abundance of glucose) disinhibits the PDC and reroutes pyruvate from gluconeogenesis to lipogenesis

5. Stoichiometry of the Krebs Cycle:
Malate leaves the mitosol and goes into gluconeogenesis
Stoichiometry of the Krebs Cycle:
CH3-CO-CoA + 3 NAD+ FAD + GDP + Pi + 2 H2O 
2 CO2 + 3 NADH + FADH2 + GTP + 2 H+ + CoA
ΔG’°=1.1 kcal/mole (sluggish)
Ac~CoA never leaves mito to favor TCA
but citrate leaves the mito. and serves as substrate for lipid synthesis
Availability of oxaloacetate (OAA) is one of the main limiting steps of TCA. The [OAA] is 1/10 of the other intermediates of TCA. Remember pyruvate carboxylase is anaplerotic. Why Acetyl-CoA activates pyruvate carboxylase?
Do you remember from glycolysis that the active metabolite (glyceraldehyde) is often kept in short supply?
The green arrow
indicates that the
equilibrium is displaced
towards malate.
Succinate dehydrogenase
is part of the mito. membrane and the respiratory chain
Isocitrate dehydrogenase is the key enzyme (committed step). Strictly dependent on the ration of ADP/ATP and NAD+/NADH. Makes TCA aerobic by the “substrate control”.
Succinyl~CoA accumulates in mitosol and serves as feedback inhibitor of citrate synthase, donor of CoA~ to activate ketone bodies and fatty acids and is also a precursor of porphyrines.
Odd carbon number fatty acids enter here and contribute to gluconeogenesis exiting the TCA as malate.
Very similar to PDC but has no intrinsic protein kinases & phosphatases. Otherwise has ~ the same regulation

6. Malonic (HOCO-CH2-COOH)
Aconitase is inhibited by fluoroacetate an enzyme activated inhibitor (often called suicidal enzyme inhibitor).
Malonic (HOCO-CH2-COOH)
acid is the archetypal example of a competitive inhibitor: it acts against succinate dehydrogenase (complex II) in the respiratory electron transport chain.
Odd carbon acids converts into propionyl-CoA which cannot directly enter either beta oxidation or the citric acid cycles. Instead it is carboxylated to D-methylmalonyl-CoA, which is isomerized to L-methylmalonyl-CoA. A vitamin B12-dependent enzyme catalyzes rearrangement of L-methylmalonyl-CoA to succinyl-CoA, which is an intermediate of the citric acid cycle.

7. Notice however, that O2 is not actually present.
The cycle is totally aerobic because of substrate (availability) control.
Notice however, that O2 is not actually present.
The * indicate the requirement of an oxidized NAD+ or FAD for the TCA to proceed. The II indicate the requirement of ADP for the cycle to proceed.

8. The yield of ATP per NADH+H+
depends on the shuttle used.

10. Beware of GLOBAL WARMING! Be nice stop breathing!
The next slide is to Integrate what you learned
in the lecture about oxidative/phosphorylation.
The objective of this slides is to account for all the moles of ATP produced per mole of glucose metabolized to CO2 and H2O.
The purpose is to get familiarized with the intricacy of interactions among metabolic pathways.
Beware of GLOBAL WARMING! Be nice stop breathing!
When you inhale feel grateful that there is still some O2 left for you to breath.
When you exhale CO2 feel guilty because you are destroying the planet. Or, maybe not, perhaps you are feeding the plants that will be the food of tomorrow.