Download Lecture 15 (Parker) - Department of Chemistry ::: CALTECH

ATP
The citric acid cycle is the final common pathway of the
oxidation of fuel molecules. Most fuel molecules enter
the pathway as Acetyl CoA:
Citric acid cycle=Krebs cycle=tricarboxylic acid (TCA)
cycle
Acetyl CoA
ATP
The citric acid cycle itself does not generate a large amount of
ATP, instead it removes electrons from Acetyl CoA forming
NADH and FADH2. These electron carriers yield nine ATP
molecules when oxidized by oxidative phosphorylation.
Electrons released in the re-oxidation of NADH and FADH2
flow through a series of membrane proteins to generate a proton
gradient across the mitochondrial membrane. These protons
then flow through ATP synthase to generate ATP from ADP.
The citric acid cycle, in conjunction with oxidative
phosphorylation provide the vast majority of energy used by
aerobic cells (>90% in human beings)
ATP
The Pyruvate Dehydrogenase Complex Links Glycolysis
to the Citric Acid Cycle and is irreversible:
Pyruvate
dehydrogenase
links glycolysis to
the citric acid cycle
ATP
Electron micrograph of the giant pyruvate dehydrogenase
complexes in E. coli
The pyruvate dehydrogenase consists of three distinct
enzymes:
The reaction requires the three enzymes of pyruvate
dehydrogenase and five coenzymes; thiamine pyrophosphate
(TPP), lipoic acid and FAD serve as catalytic cofactors:
The conversion of pyruvate to acetyl CoA consists of three
steps: decarboxlyation, oxidation and transfer of the acetyl
group to CoA
These three steps are coupled in the enzyme complex
Decarboxylation: pyruvate combines with TPP to
yield hydroxyethyl-TPP this is catalyzed by the
E1enzyme of the complex
:skip
Oxidation: the hydroxyethyl group attached to TPP is oxidized to
form an acetyl group while being simultaneously transferred to
lipoamide
Formation of Acetyl CoA: The acetyl group is transferred from
acetyllipoamide to CoA to form acetyl CoA:
Dihydrolipoyl transferase (E2) catalyzes this reaction
To complete another catalytic cycle the dihydrolipoamide is
oxidized to lipoamide:
In this fourth step the oxidized form of lipoamide is regenerated
by dihydrolipoyl dehydrogenase (E3)
Pyruvate
dehydrogenase
complex
E2 (trimer) has
the reactive
disulfide bond
of Lipoamide
tethered
through an e-N
of lysine
E2 (a3)
Reactions of the pyruvate dehydrogenase complex: 1) Decarboxylation, (2)
insertion of lipoamide arm into E1, (3) E1 catalyzes the transfer of the
acetyl group to lipoamide, (4) the acetyl moiety is transferred to CoA, (5)
E3 oxidized lipoamide by FAD, (6) NADH is produced by the re-oxidation
of FADH2
The first enzyme of the Citric Acid Cycle Citrate synthetase
forms citrate from oxaloacetate and acetyl CoA:
(condensation reaction)
Citrate synthase is first bound by oxaloacetate resulting in a
significant conformational change generating a binding site for
acetyl CoA
(homo-dimer)
Aconitase catalyzes the isomerization of citrate into isocitrate
so that the 6-carbon unit can undergo oxidative
decarboxylation:
Aconitase is a non-heme iron containing enzyme the
Fe-S cluster participates in the dehydration and rehydration of the substrate
Binding of citrate to the Fe-S complex of aconitase: only
one atom of Fe is directly involved:
Isocitrate is oxidized and decarboxylated to a-ketoglutarate by
the enzyme isocitrate dehydrogenase:
This is the first of four oxidative reduction reactions of the citric acid cycle
generating NADH
Succinyl coenzyme A is formed by the oxidative decarboxylation
of a-ketoglutarate this reaction is catalyzed by the enzyme aketoglutarate dehydrogenase complex:
a-ketoglutarate dehydrogenase complex consists of three
kinds of enzymes and is homologous to the pyruvate
dehydrogenase complex
Succinyl CoA synthetase (aka: succinate thiokinase) both ATP
and GTP can be formed from their diphospho-counterparts:
ADP
ATP
Reaction mechanism of succinyl CoA synthetase:
1) Orthophosphate displaces CoA, (2) histidine residue removes the phosphate resulting in
the release of succinate, (3)the phospho histidine residue swings over to the bound ADP
and (4) the phosphoryl group is transferred to the nucleoside diphosphate forming the
triphosphate
ADP
ATP
Structure of succinyl CoA synthetase
Oxaloacetate is regenerated by the oxidation of succinate:
Succinate
dehydrogenase
Fumarase
Oxidation-hydrationoxidation a common
metabolic motif
Malate
dehydrogenase
Fumarase catalyzes a
stereospecific trans
addition of H+ and
OH- so that only the
L-isomer of malate is
formed
Citric Acid Cycle
The citric acid cycle produces high-transfer-potential electrons,
ATP and CO2
Acetyl CoA + 3 NAD++ FAD + ADP + Pi + H2O=
2CO2 + 3NADH + FADH2 + ATP + 2H+ + CoA
ATP
The formation of
Acetyl CoA by
pyruvate
dehydrogenase is
irreversible and
therefore regulated
The citric acid cycle is controlled at several points
The rate of the citric acid cycle is precisely adjusted to meet the
ATP needs in an animal cell.
The two primary control points are the allosteric enzymes;
isocitrate dehydrogenase and a-ketoglutarate dehydrogenase
These are the first two enzymes in the cycle to generate highenergy electrons
PDH
ID
aKGD
The first control point isocitrate dehydrogenase is allosterically
stimulated by ADP, which enhances the enzyme’s affinity for
substrates.
The binding of substrate, NAD+, Mg+ and ADP are mutually
cooperative.
In contrast ATP is inhibitory as is binding of the reaction product
NADH which inhibits isocitrate dehydrogenase by displacing NAD+.
Note that several steps in the cycle require NAD+ or FAD+ which are
abundant only when the energy charge is low.
PDH
ID
aKGD
The second control site in the citric acid cycle is a-ketoglutarate
dehydrogenase.
Some aspects of this enzyme’s control are like those of the
pyruvate dehydrogenase complex, as might be expected by the
homology of the reactions catalyzed by these two enzymes.
a-ketoglutarate dehydrogenase is inhibited by the products of the
reaction succinyl CoA and NADH.
In addition a-ketoglutarate dehydrogenase is inhibited by elevated
levels of ATP.
The use of isocitrate dehydrogenase and a-ketoglutarate
dehydrogenase as control points integrate the citric acid cycle with
other metabolic pathways.
The inhibition of isocitrate dehydrogenase leads to a build up citrate
because the inter-conversion of isocitrate and citrate readily occurs.
Citrate can be transported to the cytoplasm where it signals
phosphofructokinase to reduce glycolysis.
If energy needs are met and ATP levels high the components of the
citric acid cycle can be used in other metabolic pathways:
mitochondrial
enzyme
Pyruvate
dehydrogenase
complex
Low ATP levels favor
active pathways to
produce energy
Hat makers of the past used HgNO3 to soften the firs used in hat making. The
Hg diffuses through the skin inhibiting the E2 enzyme of pyruvate
dehydrogenase through the dihydrolipoyl groups. This causes a neurological
disorder; hence the term “Mad-Hatter”
Neuronal cells are particularly susceptible to inhibitors of pyruvate
dehydrogenase because they can only use glucose as a source of
fuel. Other cell types can break down fats as a source of fuel for
the citric acid cycle.
Developed during WW I as an antidote to lewisite an arsenic based
chemical weapon (gas) BAL (British anti-lewisite) it functions to
chelate the arsenite removing it from the cell:
Defects in the citric acid cycle contribute to the development of
cancer
Three enzymes are involved: succinate dehydrogenase, fumarase
and pyruvatedehydrogenase kinase
Hypoxia inducible factor 1 (HIF-1) is a transcription factor that is
normally unstable however when stabilized by hypoxia (low O2
concentration) HIF-1 can now activate genes involved in glycolysis.
Defects in the above three enzymes can result in the accumulation
of succinate and fumarate in the mitochondria allowing
accumulation in the cytoplasm. In the cytoplasm these substrates
inhibit the enzyme required to lead to the degradation of HIF-1
(prolyl hydrolase 2) and glycolysis is favored by the action of
HIF-1.