Download Krebs cycle

Ferchmin 2016
Index:
1.
2.
3.
4.
5.
6.
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.
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?
The –SH reduces the other –S- of lipoic acid
The pyruvate dehydrogenase complex is s true multienzymatic complex
Electron micrography of
the PDC of E. coli.
Model of the PDC of E. coli.
Red: E1 pyruvate dehydrogenase
Yellow: E2 transacetylase
Green: E3 dihydrolipoyl dehydrogenase
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
energy
2) Lipoate
transacetylase
(E2 )
Lipoic acid
Dehydrogenation of hydroxyethyl and
transfer of acetyl to CoA.
60 subunits
CoA
TCA
substrate
CoA~Acetyl
TCA substrate
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;
Reducing
power
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
The regulatory subunits of the PDC are intrinsic to the complex
Ca2+ in muscle &
insulin in liver
Insulin (means abundance of glucose) disinhibits the PDC and
reroutes pyruvate from gluconeogenesis to lipogenesis
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
The green arrow
indicates that the
equilibrium is displaced
towards malate.
Succinate dehydrogenase
is part of the mito. membrane
and the respiratory chain
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 AcetylCoA activates pyruvate carboxylase?
Do you remember from glycolysis that the active
metabolite (glyceraldehyde) is often kept in short
supply?
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”.
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
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.
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 propionylCoA 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-methylmalonylCoA. A vitamin B12-dependent enzyme
catalyzes rearrangement of Lmethylmalonyl-CoA to succinyl-CoA, which
is an intermediate of the citric acid cycle.
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.
The yield of ATP per
NADH+H+
depends on the
shuttle used.
The next slide is for you to review after you had the lecture about
oxidative/phosphorylation.
The objective 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
ATP YIELD FROM THE COMPLETE OXIDATION OF GLUCOSE
Reaction sequence
ATP yield per glucose
Glycolysis: glucose into pyruvate (in the cytosol)
Phosphorylation of glucose
Phosphorylation of fructose 6-phosphate
Dephosphorylation of 2 molecules of 1,3-DPG
Dephosphorylation of 2 molecules of phosphoenolpyruvate
-1
-1
+2
+2
2 NADH are formed in the oxidation of 2 molecules of *
glyceraldehyde 3-phosphate (Shuttle to mitochondria and oxidative phosphorylation)
Conversion of pyruvate into acetyl CoA (inside mitochondria)
2 NADH are formed
**
Citric acid cycle (inside mitochondria)
2 molecules of guanosine triphosphate are formed from
2 molecules of succinyl CoA
6 NADH are formed in the oxidation of 2 molecules of each
isocitrate, α-ketoglutarate, and malate
2 FADH2 are formed in the oxidation of 2 molecules of 
succinate
+2
Oxidative phosphorylation (inside mitochondria)
2 NADH formed in glycolysis; each yields 2 ATP
(Assuming transport of NADH by the glycerol phosphate
shuttle)
2 NADH formed in the oxidative decarboxylation of pyruvate:
each yields 3 ATP
2 FADH2 formed in the citric acid cycle;
each yields 2 ATP
6 NADH formed in the citric acid cycle;
each yields 3 ATP
+4 *
+6 **
+4
+18 
+36 or 38
These 36 ATP could be 38 if the malate aspartate shuttle is used.
However, in real life, the mitochondrion uses part of its H+ gradient for
“housekeeping” and the approximate yield of ATP/mol of glucose is roughly 24.