Transcript Further Details of Mechanism

The Citric Acid Cycle Can
Be a Multistep Catalyst
• Oxaloacetate is regenerated
• The cycle is a mechanism for oxidizing acetyl
CoA to CO2 by NAD+ and Q
• The cycle itself is not a pathway for a net
degradation of any cycle intermediates
• Cycle intermediates can be shared with other
pathways, which may lead to a resupply or net
decrease in cycle intermediates
1. Citrate Synthase
• Citrate formed from acetyl CoA and oxaloacetate
• Only cycle reaction with C-C bond formation
Proposed mechanism of citrate synthase
Citrate synthase generates a new C-C bond using
acetyl CoA in a Claisen condensation
1. The enolate nucleophile is generated by “push-pull” acid/base catalysis
2. The enolate attacks the -keto position of oxaloacetate in a reversible aldol reaction
3. Hydrolysis of the thioester, also catalyzed by Asp375 and His274, is exothermic and
essentially irreversible. This drives the reaction to completion.
2. Aconitase
• Elimination of H2O from citrate to form C=C
bond of cis-aconitate
• Stereospecific addition of H2O to cis-aconitate
to form 2R,3S-Isocitrate
Reaction of Aconitase
3. Isocitrate Dehydrogenase
• Oxidative decarboxylation of isocitrate to
-ketoglutarate (-kg) (a metabolically irreversible
reaction)
• One of four oxidation-reduction reactions of the cycle
• Hydride ion from the C-2 of isocitrate is transferred to
NAD+ to form NADH
• Oxalosuccinate is decarboxylated to -kg
Spontaneous CO2 production can occur with a -keto acid
-keto acids are unstable in protic solvents
• They will pick up a proton on the ketone oxygen and this triggers
decarboxylation.
• Generation of CO2 makes this reaction irreversible and highly exothermic.
• How do you capture the energy from this process?
• In biochemistry, the energy of “combustion” is partially harvested by
coupling most decarboxylation reactions to an oxidation reaction.
Isocitrate dehydrogenase reaction
Aconitase uses an FeS cluster as a Lewis acid
catalyst
Isocitrate dehydrogenase captures the energy of
decarboxylation
•The oxidation of an alcohol to a ketone in oxalosuccinate is reversible
and near equilibrium.
•Lewis acid catalyzed decarboxylation of the -keto acid drives this
reaction to 100% completion.
•Energy of decarboxylation is “captured” in the reduced NADH through
the coupling of these two reactions on the same enzyme.
The Citric Acid Cycle
Oxidizes AcetylCoA
• Table 12.2
4. The a-Ketoglutarate Dehydrogenase
Complex
Structure of a-Ketoglutarate
dehydrogenase complex
• Similar to pyruvate dehydrogenase complex
• Same coenzymes, identical mechanisms
E1 - -ketoglutarate dehydrogenase (with TPP)
E2 - succinyltransferase (with flexible lipoamide
prosthetic group)
E3 - dihydrolipoamide dehydrogenase (with FAD)
-ketoglutarate dehydrogenase is is a thiamine enzyme
similar to pyruvate dehydrogenase
 -ketoglutarate dehydrogenase is a large protein
complex that shares some identical subunits with
the pyruvate dehydrogenase complex
• Decarboxylation requires thiamine, lipoic acid, and
generates electrons that reduce NAD+ to NADH
Succinyl CoA Synthetase runs backwards to make GTP
GTP can
be used to
make ATP
in another
enzyme
Thioester is used to make a
phosphoanhydride bond
Phosphohistidine is high energy
attacked to make a phosphodiester bond
6. The Succinate
Dehydrogenase (SDH) Complex
• Located on the inner mitochondrial membrane
(other components are dissolved in the matrix)
• Dehydrogenation is stereospecific; only the
trans isomer is formed
• Substrate analog malonate is a competitive
inhibitor of the SDH complex
Reaction of the succinate
dehydrogenase complex
(a) Ubiquinone,
(b) Plastoquinone
• Hydrophobic tail of each is composed of 6 to 10
five-carbon isoprenoid units
• The isoprenoid chain allows these quinones to
dissolve in lipid membranes
• Three oxidation states of
ubiquinone
• Ubiquinone is reduced in
two one-electron steps
via a semiquinone free
radical intermediate.
Reactive center is shown
in red.
Succinate dehydrogenase contains an
FAD cofactor
Eh′ = -0.040 V (NAD+ is -0.320 V)
7. Fumarase
• Stereospecific trans addition of water to the
double bond of fumarate to form L-malate
Fumarase catalyzes hydration of an alkene
Class II fumarase (human and yeast) uses acid/base chemistry to catalyze
trans addition of water via a carbanion intermediate
Histidine
Lysine
Class I fumarase (bacteria) uses an iron-sulfur cluster to catalyze the same
chemistry. It has not been studied experimentally, but it is assumed to work
by a mechanism similar to aconitase.
8. Malate Dehydrogenase
Reduced Coenzymes Fuel the
Production of ATP
• Each acetyl CoA entering the cycle nets:
(1) 3 NADH
(2) 1 QH2
(3) 1 GTP (or 1 ATP)
• Oxidation of each NADH yields 2.5 ATP
• Oxidation of each QH2 yields 1.5 ATP
• Complete oxidation of 1 acetyl CoA = 10 ATP
Glucose degradation via glycolysis, citric
acid cycle, and oxidative phosphorylation
Three point attachment of
prochiral substrates to enzymes
• Chemically identical groups a1 and a2 of a prochiral
molecule can be distinguished by the enzyme
• Fates of carbon
atoms in the cycle
• Carbon atoms from
acetyl CoA (red) are
not lost in the first
turn of the cycle
Reducing Power
• Electrons of reduced coenzymes flow toward O2
• This produces a proton flow and a transmembrane
potential
• Oxidative phosphorylation is the process by
which the potential is coupled to the reaction:
ADP + Pi
ATP
Reduced Coenzymes Conserve Energy
from Biological Oxidations
• Amino acids, monosaccharides and lipids are
oxidized in the catabolic pathways
• Oxidizing agent - accepts electrons, is reduced
• Reducing agent - loses electrons, is oxidized
• Oxidation of one molecule must be coupled with
the reduction of another molecule
Ared + Box
Aox + Bred
Diagram of an electrochemical cell
• Electrons flow
through external
circuit from Zn
electrode to the
Cu electrode
Reduction Potentials
Cathode (Reduction)
Half-Reaction
Li+(aq) + e- -> Li(s)
K+(aq) + e- -> K(s)
Ca2+(aq) + 2e- -> Ca(s)
Na+(aq) + e- -> Na(s)
Zn2+(aq) + 2e- -> Zn(s)
Cu2+(aq) + 2e- -> Cu(s)
O3(g) + 2H+(aq) + 2e- -> O2(g) + H2O(l)
F2(g) + 2e- -> 2F-(aq)
Standard Potential
E° (volts)
-3.04
-2.92
-2.76
-2.71
-0.76
0.34
2.07
2.87
Standard reduction potentials
and free energy
• Relationship between standard free-energy
change and the standard reduction potential:
DGo’ = -nFDEo’
n = # electrons transferred
F = Faraday constant (96.48 kJ V-1)
DEo’ = Eo’electron acceptor - Eo’electron donor
Example
Suppose we had the following voltaic cell at 25o C:
Cu(s)/Cu+2 (1.0 M) // Ag+(1.0 M)/ Ag (s)
What would be the cell potential under these conditions?
Example
Suppose we had the following voltaic cell at 25o C:
Cu(s)/Cu+2 (1.0 M) // Ag+(1.0 M)/ Ag (s)
What would be the cell potential under these conditions?
Ag+ + e- ---> Ag0
Cu+2 + 2e- ----> Cu0
E0red = + 0.80 v
E0red = + 0.337 v
Example: Biological Systems
Both NAD+ and FAD are oxidizing agents
The question is which would oxidize which?
OR
Which one of the above
is the spontaneous reaction?
in which DG is negative
To be able to answer the question
We must look into the “electron donation”
capabilities of NADH and FADH2
i.e. reduction potentials of
NADH and FADH2
Remember,
DEo’ = Eo’electron acceptor - Eo’electron donor
For a spontaneous reaction DEo ’ must be positive
Therefore,
rearrange
Add the two reactions
electron
acceptor
electron
donor
Regulation of the Citric Acid Cycle
• Pathway controlled by:
(1) Allosteric modulators
(2) Covalent modification of cycle enzymes
(3) Supply of acetyl CoA
(4) Regulation of pyruvate dehydrogenase
complex controls acetyl CoA supply
Regulation of the PDH complex
• Increased levels of acetyl CoA and
NADH inhibit E2, E3 in mammals and E. coli
Regulation of mammalian PDH
complex by covalent modification
• Phosphorylation/dephosphorylation of E1
Further regulation of the PDH complex
Pyruvate dehydrogenase kinase (PDK)
• PDK is activated by NADH and acetyl CoA
(leads to inactivation of the PDH complex)
• PDK is inhibited by pyruvate and ADP (leads to
activation of the PDH complex)
Pyruvate dehydrogenase phosphatase (PDP)
• PDP activity is stimulated by Ca2+ (leads to an
activation of the PDH complex)
Control points in the citric acid cycle
Rate is adjusted to meet the cell’s
need for ATP. Three allosteric
enzyme control points:
PDH - inhibited by NADH, acetyl
CoA, and ATP.
Isocitrate dehydrogenase stimulated by ADP; inhibited by ATP
and NADH
a-ketoglutarate dehydrogenase—
inhibited by NADH, succinyl CoA,
high energy charge.
The Glyoxylate Cycle
• Pathway for the formation of glucose from
noncarbohydrate precursors in plants, bacteria
and yeast (not animals)
• Glyoxylate cycle leads from 2-carbon
compounds to glucose
• In animals, acetyl CoA is not a carbon source for
the net formation of glucose (2 carbons of acetyl
CoA enter cycle, 2 are released as 2 CO2)
Glyoxylate cycle - formation of glucose
• Formation of glucose from acetyl CoA (or any
substrate that is a precursor to acetyl CoA)
• Ethanol or acetate can be metabolized to
acetyl CoA and then to glucose via the
glyoxylate cycle
• Stored seed oils in plants are converted to
carbohydrates during germination
Isocitrate lyase: first bypass
enzyme of glyoxylate
Malate synthase: second bypass
enzyme of glyoxylate
Glyoxylate cycle in germinating
castor beans
• Conversion of acetyl CoA to glucose
requires the transfer of metabolites among
three metabolic compartments
(1) The glyoxysome
(2) The cytosol
(3) The mitochondrion