4. TCA Cycle
Krebs cycle
Citric Acid Cycle
Central catabolic pathway
TCA cycle is the final common pathway
of th oxidation of acetyl CoA, formed
from carbohydrates, fatty acids and
amino acids.
Operates under aerobic conditions only
5. Pyruvate dehydrogenase complex
Strictly speaking this reaction is not a part
of the TCA cycle.
A bridge between the Glycolysis and TCA
cycle
Pyruvate dehydrogenase complex (PDC) is a
multienzyme complex of three enzymes that
transform pyruvate into acetyl-CoA by a
process called oxidative decarboxylation.
Occurs in the mitochondrial matrix
links the glycolysis to the citric acid cycle
6. The pyruvate is transported into the
mitochondria via the pyruvate carrier, an
antiporter, in exchange for a hydroxide ion
(OH-).
7. Structure & function of PDH
Located in the mitochondrial matrix of
eukaryotes
Multienzyme complex greatly increases
the efficiency.
Irreversible reaction
consists of a total of 60 subunits
Mol Wt. 9X106
organized into three functional
proteins
9. Pyruvate dehydrogenase (E1)
Initially, pyruvate and thiamine
pyrophosphate (TPP) are bound by pyruvate
dehydrogenase subunits.
The thiazolium ring of TPP is in a zwitterionic
form, and the anionic C2 carbon performs a
nucleophilic attack on the C2 (ketone)
carbonyl of pyruvate.
The resulting hemithioacetal undergoes
Decarboxylation to produce an acyl anion
equivalent
This anion attacks S1 of an oxidized lipoate
species that is attached to a lysine residue.
10. Pyruvate dehydrogenase (E1)
In a ring-opening SN2-like mechanism, S2 is
displaced as a sulfide or sulfhydryl moiety.
Subsequent collapse of the tetrahedral
hemithioacetal ejects thiazole, releasing the
TPP cofactor and generating a thioacetate on
S1 of lipoate.
The E1-catalyzed process is the rate-limiting
one of the whole pyruvate dehydrogenase
complex.
11. Dihydrolipoyl transacetylase
(E2)
At this point, the lipoate-thioester
functionality is translocated into the
dihydrolipoyl transacetylase (E2)
active site, where a transacylation
reaction transfers the acetyl from the
"swinging arm" of lipoyl to the thiol of
coenzyme A.
This produces acetyl-CoA, which is
released from the enzyme complex and
subsequently enters the citric acid
cycle.
12. Dihydrolipoyl dehydrogenase (E3)
The dihydrolipoate, still bound to a lysine
residue of the complex, then migrates to the
dihydrolipoyl dehydrogenase (E3) active site
where it undergoes a flavin-mediated
oxidation, identical in chemistry to disulfide
isomerase.
First, FAD oxidizes dihydrolipoate back to its
lipoate resting state, producing FADH2.
Then, a NAD+ cofactor oxidizes FADH2 back
to its FAD resting state, producing NADH.
13.
14.
15.
16.
17. Lipoate acts as
both electron and
acyl carriers.
It swings
between the three
different active
sites of the
pyruvate
dehydrogenase
complex.
18.
19.
20.
21.
22.
23.
24.
25. The Chemical Logic of TCA
Understand this!
TCA seems like a complicated way to
oxidize acetate units to CO2
But normal ways to cleave C-C bonds and
oxidize don't work for CO2:
cleavage between Cs and to a
carbonyl
an -cleavage of an -hydroxyketone
26. The Chemical Logic of TCA
A better way to cleave acetate...
Better to condense acetate with
oxaloacetate and carry out a -
cleavage - TCA combines this with
oxidation to form CO2, regenerate
oxaloacetate and capture all the
energy as NADH and ATP!
27. Citrate Synthase
Nonproductive hydrolysis of the acetyl CoA is
prevented because citrate synthase binds
oxaloacetate first.
Once oxaloacetate binds, the enzyme undergoes a
conformational change, closing the oxaloacetate
binding site and opening the acetyl CoA binding site.
This means that when the acetyl CoA binds,
oxaloacetate is ready and positioned for
catalysis, limiting the nonproductive hydrolysis of
acetyl CoA.
Additionally, amino acid side chains necessary for
hydrolysis are not in ready position until
citryl CoA, the intermediate, is formed. This, too,
prevents nonproductive hydrolysis.
31. Aconitase
The hydroxyl group of citrate is not
properly positioned for the
decarboxylation steps, hence Citrate
is a poor substrate for oxidation
Therefore, citrate undergoes an
isomerization catalyzed by aconitase
Aconitase uses an iron-sulfur cluster
32.
33.
34. Isocitrate Dehydrogenase
The oxidative decarboxylation of
isocitrate is the first of four
oxidation-reduction reactions.
Requires one molecule of NAD+.
Releases one molecule of CO2 and
one molecule of NADH.
The intermediate, oxalosuccinate, is
unstable and readily loses CO2.
35.
36.
37.
38. α-Ketoglutarate
Dehydrogenase Complex
The oxidative decarboxylation of α -
ketoglutarate to succinyl CoA is the
second of four oxidation-reduction
reactions.
Requires one molecule of NAD+ and
one molecule of CoA.
Releases one molecule of CO2 and
one molecule of NADH.
39. The α-ketoglutarate dehydrogenase complex
is homologous to the pyruvate dehydrogenase
complex with the reactions being very similar.
In fact, the E3 component is not homologous,
but the same enzyme.
1. α-ketoglutarate dehydrogenase
2. Dihydrolipoyl transsuccinylase
3. Dihydrolipoyl dehydrogenase (identical to PDC)
Five coenzymes used -
TPP, CoA-SH, Lipoic acid, NAD+, FAD
40.
41. Succinyl CoA Synthetase
The hydrolysis of the thioester bond in succinyl CoA
releases an amount of energy comparable to that of
ATP.
Releases one molecule of CoA and
one molecule of GTP.
The hydrolysis of the thioester is coupled to the
production of GTP.
This is the only step in the citric acid cycle in which a
compound with a high phosphoryl transfer potential
(GTP) is produced through substrate-level
phosphorylation (the phosphoryl group comes from a
substrate with a high phosphoryl transfer potential as
opposed to an orthophosphate molecule).
GTP can transfer its γ-phosphoryl group to ADP in a
reaction catalyzed by nucleoside disphosphokinase.
42.
43.
44. Succinate Dehydrogenase
Catalyzes the third of four oxidation-
reduction steps, the oxidation of
succinate to fumarate.
The hydrogen acceptor is FAD as
opposed to NAD+.
FAD is usually the hydrogen acceptor
when there are two hydrogen atoms to
be transferred.
Releases one molecule of FADH2.
45. Succinate dehydrogenase
Succinate dehydrogenase, unlike the other
enzymes of the citric acid cycle, is embedded
in the mitochondrial membrane.
Unlike NADH produced by other reactions,
FADH2 in the oxidation of succinate does
not dissociate from the enzyme. Instead, the
two electrons are transferred directly to
iron-sulfur clusters in succinate
dehydrogenase.
The electrons transferred to the iron-sulfur
clusters continue forward in the electron
49. Malate Dehydrogenase
Catalyzes the fourth of four
oxidation-reduction steps, the
oxidation of malate to oxaloacetate.
Requires one molecule of NAD+.
Releases one molecule of NADH.
50.
51. The Net Reaction of the Citric
Acid Cycle
acetyl CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O
2 CO2 + 3 NADH + FADH2 + GTP + 2H+ + CoA
Summary
1. Two carbon atoms enter the cycle as the acetyl
group of acetyl CoA. Two carbon atoms leave the cycle
as CO2 in two decarboxylation steps.
2. Four pairs of hydrogen atoms leave the cycle in four
oxidations reactions. Three NAD+ are reduced to
NADH, one FAD is reduced to FADH2,
3. One high-phosphoryl-potential molecule is
produced—GTP.
4. Two molecules of H2O are consumed.
The NADH and FADH2 formed in the citric acid cycle
lose their electrons in the electrontransport chain,
which ultimately leads to the generation of ATP.
62. 10. Anaplerotic Role of TCA Cycle
Pyruvate carboxylase - converts pyruvate to
oxaloacetate (in animals), is activated by
acetyl-CoA
PEP carboxylase - converts PEP to
oxaloacetate inhibited by aspartate
Malic enzyme converts pyruvate into malate
Transaminases convert Glutamate to alpha
keto glutarate & Aspatrate to oxaloacetate.
PEP carboxykinase - could have been an
anaplerotic reaction.
CO2 binds weakly to the enzyme, but
oxaloacetate binds tightly, so the reaction
favors formation of PEP from oxaloacetate
63.
64.
65. Glycolysis
Reaction Energy
Product
factor ATP Equivalents
(@2.5 ATP/NAD)
ATP Equivalents
(@3 ATP/NAD)
Hexokinase ADP 1 x -1 - 1 -1
PFK ADP 1 x-1 - 1 -1
GA-3-P DH NADH 2 x 2.5
(1.5)*
5 (3)* 6 (4)*
PGA Kinase ATP 2 x1 2 2
Pyruvate Kinase ATP 2 x 1 2 2
66. Pyruvate DH Complex & Kreb's Cycle
Reaction
Energy
Product
factor
ATP
Equivalents
(@2.5
ATP/NAD)
ATP
Equivalents
(@3
ATP/NAD)
Pyruvate DH
Complex
NADH 2 x 2.5 5 6
Isocitrate DH NADH 2 x 2.5 5 6
2-oxoglutarate DH
Complex
NADH 2 x 2.5 5 6
Succinyl-CoA
Synthetase
GTP 2 x 1 2 2
Succinate DH FADH2 2 x 1.5 3 4
Malate DH NADH 2 x 2.5 5 6
TOTAL= 32 (30)* 38 (36)*
* In some tissues (insect flight muscle, fast twitch muscle) the reducing
equivalents of NADH must be pumped against a gradient at a cost of 1 ATP (it is
used to make FADH2).
67.
68.
69. Inhibitots of TCA Cycle
Aconotase inhibited by Fouroacetate
(Non Competitive Inhibition)
α-Ketoglutarate Dehydrogenase
Complex is inhibited by Arsentie.
(Non Competitive Inhibition)
Succinate Dehydrogenase inhibited by
Malonate (Competitive Inhibition)