The Citric Acid Cycle - Biochemistry - Lecture Notes, Study notes for Biochemistry. Biyani Girls College

Biochemistry

Description: The Citric Acid Cycle, Citrate Synthase, Isocitrate Dehydrogenase, Pyruvate Dehydrogenase, Dihydrolipoyl Transacetylase, Dihydrolipoyl Dehydrogenase, Succinate Dehydrogenase, Fumarase Catalyzes, Malate Dehydrogenase, Net Reaction of the Citric Acid Cycle are few points from this lecture notes. If you are looking for complete set of lectures on biochemistry, you can find it in my documents section.
Showing pages  1  -  4  of  6
The Citric Acid Cycle
I. Citrate Synthase
The first reaction of the citric acid cycle is the condensation of acetyl-CoA
and oxaloacetate to form citrate and CoA-SH.
The enzyme that catalyzes this reaction is called citrate synthase.
Go’ = -32.2 kJ/mol
The change in free energy based on the steady state concentrations of
oxaloacetate, acetyl-CoA and citrate in the mitochondria of isolated from pig
hearts is:
G = -53.9 kJ/mol
A very exergonic reaction and irreversible.
The mechanism of citrate synthase is
shown to the left. In the active site of the
enzyme we have two histidines and an
aspartate which function as general acids
and bases during catalysis.
The first step is to generate an enol of
acetyl-CoA. Asp-375 functions as a
general base abstracting a proton for the
methyl group of Acetyl-CoA. His-274
concertedly functions as a general acid
donating a proton to the carbonyl to form
the enol.
The enol generated in the first step is
converted into a nucleophile by the
abstraction of the enol hydrogen by His-
274 now functioning as a general base.
The electrons of the double bond attack the
electrophilic center of the ketone of
oxaloactete. His-320 functions in concert
as a general acid donating its proton to the
carbonyl oxygen of oxaloacetate to form
citryl-CoA. Citryl-CoA spontaneously
hydrolyses while it is still bound to the
active site to generate coenzyme A and
citrate.
H3CC
O
SCoA
CO2-
CO
CH2
CO2-
CH2
CCO
2-
CH2
CO2-
CO2-
HO
+
CoASH + H+
CO2
-
O
H2C
-O2C
His274
CH2
N
NH
H
+C
C
O
CoAS
H
H
H
O
-O
Asp375
CO2
-
O
H2C
-O2C
His274
CH2
N
NH
HC
C
O
CoAS
H
H
H
O
O
Asp375
His320
CH2
N
NH
H
+
CO2
-
OH
H2C
-O2C
CH2
C
O
CoAS
His320
CH2
N
NH
H
O
O
Asp375
His274
CH2
N
NH
H
H2O
CH2
CCO
2
-
CH2
CO2
-
CO2
-
HO
CoASH + H+
Docsity.com
Citrate synthase is a homodimer with symmetry as you can see to the left.
It has a sequential order kinetic mechanism.
First the enzyme binds oxaloacetate which induces the large conformational
change shown (b). This is yet another example of induced fit. The
conformation change induced by oxaloacetate binding creates the acetyl-
CoA binding site and seals oxaloacetate form the aqueous solvent.
Now that you are no longer novices regarding metabolic strategies, you
probably already have a sense that citrate synthase is going be allosterically
regulated.
It is the first step of a metabolic pathway.
It catalyzes an irreversible step in the pathway.
It is a homodimer with symmetry.
And your intuitions are correct. This enzyme is a site for allosteric regulation.
One of the products of the citric acid cycle is NADH which allosterically inhibits the enzyme. Succinyl-
CoA, the product of the fifth step of the cycle also inhibits.
II. Aconitase
Citrate is a tertiary alcohol. It is difficult to oxidize tertiary alcohols because forming the
ketone would involve breaking a carbon-carbon bond. To get around this problem, citrate
is isomerized into isocitrate. Isocitrate is a secondary alcohol which can be easily
oxidized to the ketone by NAD+. The enzyme that catalyzes this migration of a hydroxyl
group is aconitase. This enzyme catalyzes the dehydration of citrate of form cis-aconitate
and then rehydrating the double bond to form isocitrate.
Go’ = +6.7 kJ/mol
The change in free energy based on the steady state concentrations of citrate and isocitrate
in the mitochondria of isolated from pig hearts is:
G = +0.8 kJ/mol ; near equilibrium.
Aconitase is an iron sulfur protein.
It contains four iron atoms complexed to four inorganic
sulfides and three cysteine sulfur atoms called a 4Fe-4S
iron- sulfur cluster. One of the iron atoms has an open
coordination site that complexes with the carboxylate
group of C3 and the hydroxyl group of citrate. This
iron residue facilitates the dehydration and rehydration
reaction and accounts for the stereospecifity of the
reaction.
CH2
CCO
2-
CH2
CO2-
CO2-
HO
C
CH
CH2
CO2-
CO2-
OHH
-O2C
C
C
H2C
CO2-
CO2-
H
CO2-
Docsity.com
III. Isocitrate Dehydrogenase
This is the first oxidation step of the pathway.
Isocitrate dehydrogenase catalyzes the oxidation of isocitrate to
oxalosuccinate which is a β−keto acid. This β−keto acid
spontaneously decarboxylates to form α−ketoglutarate. This is the
second time we have oxidized a compound to form a β−keto acid
which spontaneously decarboxylated itself. In the pentose phosphate
pathway,6-phosphogluconate dehydrogenase oxidized 6-
phosphogluconate to 3-keto-6-phosphogluconate, a β−keto acid,
which spontaneously decarboxylated to form ribulose-5-phosphate.
Go’ = -8.4 kJ/mol
The change in free energy based on the steady state concentrations of
isocitrate and α−ketoglutarate in the mitochondria of isolated from
pig hearts is:
G = -17.5 kJ/mol ;
A very exergonic, irreversible reaction.
As you expect, this irreversible reaction is allosterically regulated.
NADH and ATP are allosteric inhibitors.
ADP is an allosteric activator which lowers the Km for isocitrate by a
factor of 10. When the concentration of ADP is low, the Km for citrate is well above the physiological
concentration making the enzyme essentially inactive. With ADP bound in the allosteric binding site, the
Km is lowered by a factor of ten making the enzyme active at physiological concentrations.
IV. α−Ketoglutarate Dehydrogenase
The next step in the TCA cycle is the oxidative decarboxylation of
α−ketoglutarate to form succinyl-CoA and NADH.
Go’ = -30.0 kJ/mol
The change in free energy based on the steady state concentrations of
α−ketoglutarate and succinyl-CoA in the mitochondria of isolated from pig
hearts is:
G = -43.9 kJ/mol ;
A very exergonic, irreversible reaction
If you look at the chemical transformation involved, you will note the similarity
between this enzymatic reaction and the one catalyzed by pyruvate
dehydrogenase.
Pryruvate dehydrogenase:
Pyruvate + CoA + NAD+ acetyl-CoA + CO2 + NADH
α−Ketoglutarate dehydrogenase:
α−ketoglutarate + CoA + NAD+ succinyl-CoA + CO2 + NADH
C
CH
CH2
CO2-
CO2-
OHH
-O2C
C
CH2
CH2
CO2-
CO2-
O
NAD+
NADH + H+
CO2
C
CH
CH2
CO2-
CO2-
O
C
O
-O
C
CH
CH2
CO2-
CO2-
O-
BH
C
CH2
CH2
CO2-
CO2-
O
C
CH2
CH2
CO2-
SCoA
O
HS-CoA + NAD+
CO2 + NADH
Docsity.com
Both of these reactions include the decarboxylation of an α-keto acid and the subsequent formation of an
high energy thioester linkage with coenzyme A.
α−Ketoglutarate dehydrogenase is a humongous multienzyme complex that is very similar to the pyruvate
dehydrogenase multienzyme complex.
Pyruvate Dehydrogenase Complex of E. coli.
Enzyme
# Prosthetic
Groups
Reaction Catalyzed.
Pyruvate Dehydrogenase E1 24 TPP Oxidative Decarboxylation
Dihydrolipoyl transacetylase E2 24 Lipoamide Transfer of acetyl group
Dihydrolipoyl Dehydrogenase E3 12 FAD Regenerate lipoamide.
α-Ketoglutarate Dehydrogenase Complex of E. coli.
Enzyme
# Prosthetic
Groups
Reaction Catalyzed.
α-Ketoglutarate Dehydrogenase E1 24 TPP Oxidative Decarboxylation
Dihydrolipoyl transacetylase E2 24 Lipoamide Transfer of succinyl group
Dihydrolipoyl Dehydrogenase E3 12 FAD Regenerate lipoamide.
The mechanism for α−Ketoglutarate dehydrogenase is identical to that of pyruvate dehydrogenase.
V. Succinyl-CoA Synthetase
So far we have generated 2 molecules of CO2 and 2 molecules of NADH. The electrons of NADH will be
routed through the electron transport chain and ultimately generated 2.5 equivalents of ATP by oxidative
phosphorylation. The succinyl-CoA contains a high energy bond which is going to be utilized in this step
of the cycle.
The enzyme succinyl-CoA synthetase couples the conversion of succinyl-CoA
into succinate with the synthesis of GTP from GDP and Pi. The standard free
energy change for hydrolyzing succinyl-CoA into succinate and coenzyme A is
-33.8 kJ/mole. The standard free energy required to synthesize GTP from GDP
and Pi is +30.5 kJ/mole. If we couple these two reactions together than the
standard free energy change is -3.3 kJ/mole. This enzyme catalyzes a substrate
level phosphorylation to generate the only NTP produced directly in the citric
acid cycle. The GTP produced is converted into ATP by the enzymatic activity
of nucleoside diphosphate kinase:
ADP + GTP ATP + GDP
For succinyl-CoA synthetase:
Go’ = -3.3 kJ/mol
The change in free energy based on the steady state concentrations of all the
reactants and products in the mitochondria of isolated from pig hearts is:
G 0;
A near equilibrium reaction.
C
CH2
CH2
CO2-
SCo
A
O
CO2-
CH2
CH2
CO2-
GDP + Pi
GTP + HSCoA
Docsity.com
The preview of this document ends here! Please or to read the full document or to download it.
Document information
Embed this document:
Docsity is not optimized for the browser you're using. In order to have a better experience please switch to Google Chrome, Firefox, Internet Explorer 9+ or Safari! Download Google Chrome