Download Lehninger Principles of Biochemistry 5/e

David L. Nelson and Michael M. Cox
LEHNINGER
PRINCIPLES OF BIOCHEMISTRY
Fifth Edition
CHAPTER 16
The Citric Acid Cycle
© 2008 W. H. Freeman and Company
Cellular Respiration occurs in
three stage
- Organic fuel molecules are
oxidized to yield twocarbon fragemnts in the
form of acetyla –coA
- The acetyl group is
oxidized into carbon
dioxide in the citric acid
cycle; energy released is
conserved in the NADH
and FADH2
- This reduced coenzyme
transferred electron to
oxygen through ETS and
oxidative phosphorylation
Pyruvate is oxidized to acetyl-coA and CO2
 Oxidative decarboxylation by PDH complex
The PDH complex requires five coenzyme
TPP, FAD, NAD, CoA, lipoate
Reactive thiol, acyl carrier, thioester ( high acyl group
transfer poteintial and donate their acyl group to
acceptor molecule)
Two thiol groups, ser
both as an electron carrier
and an acyl carrier
1. PHD complex contains three
enzyme
- pyruvate DH (E1)
- dihydrolipoyl transacetylase
(E2)
- dihydrolipoyl DH (E3)
2. E2 is the point of connection
for the lipoate, attached to Lys.
3. The active site of E1 has
bound TPP
4. E3 has bound FAD
E2 has three functionally distinct domains
Intermediates never leave the enzyme surface
Step 1. : C-1 is released as CO2, C-2 is attached to TPP as a
hydroxyethyl group
Step 2. : this hydroxyethyl group is oxidized into acetate and
electrons reduced disulfide bond and then acetyl moiety
produced thioester bond
Step 3: form acetyl-CoA
Step 4 and 5: electron
transfer to NAD
1. Central to the mechanism are the winging lipoyllysyl arms
of E2, which accept from E1 the two electrons and the
acetyl group, passing them to E3
2. Substrate Channeling: allowing the intermediates to react
quickly without diffusing away from the surface of
enzyme complex
 Thus, local concentration of substrate is kept high
1.
In each turn of the cycle, on
acetyl froup enters as
acetyl-CoA and two Co2
leave; 1 OAA used and 1
OAA generated; NADH
and FADH2, GTP or ATP
2. Four or five carbon
intermediate serve as
precursor of biomolecule
3. In eucaryotes, cycle takes
place in mitochondria
- the site of most energyyielding oxidative reactions
and of the coupled
synthesis of ATP
3. In procaryotes. cycle are in
the cytosol, plasma memb.
plays a role analogues to
that of inner mitrochondrial
memb. in ATP synthesis
1.
-
Formation of citrate: the condensation of acetyl-CoA with
oxaloacetate.
Methyl carbon of acetyl group is joined to the carbonyl group (C-2) of
OAA
The hydrolysis of high-energy thioester makes the forward reaction
Citrate sythase : homodimeric enzyme; induce conformational change
by OAA binding, creating binding site for acetyl-CoA; when citroylCoA has formed , another conformational change brings about
thioester hysorlysis; ordered bisubstrate mechanism
2. Formation of isocitrate via cis-aconitate
- The aconitase catalyzes the reversible transformation of citrate to
isocitrate
- Because isocitrate is rapidly consumed in the next step of cycle, the
reaction is pulled to the right
- Iron sulfur center: binding site of the substrate, catalytic addition or
removal of water
3. Oxidation of isocitrate to a-ketoglutarate and carbon dioxide
- Isocitrate dehydrogenase catalyzed oxidative decarboxylation
- Mn2+ in active site: stabilized intermediates
4. Oxidation of a-ketoglutarate to succinyl-CoA and carbon dioxide
- a-ketoglutarate dehydrogenase complex: NAD serve as an electron
aceeptor and CoA as the carrier of the succinyl group.
- Resemble to pyruvate dehydrogenase complex:E1, E2, E3 complex.
TPP. Lipate, FAD, NAD, CoA
- Energy of oxidation is conserved in the formation of thioester bond
5. Conversion of succinyl-CoA to succinate
- Succinyl-CoA sythetase
- Thioester bond  phophoanhydride bond of GTP or ATP
6. Oxidation of succinat to fumarate
- Succinate dehydrogenase: flavoprotein, bind to the mitochondrial
inner membrane, three different iron-sulfur cluster and FAD
- Electorn pass from succinate through the FAD and iron-sulfur center
before entering ETS
Malonate is a competitive inhibitor of succinate dehydrogenase and block
the citric acid cycle
6. Hydration of fumarate to malate
6. Oxidation of Malate to Oxalocetate
- The equilibrium of this reaction less far to the left under standard
condition, but in intact cells oxaloacetate is continually remove by the
highly exergonic citrate synthase reaction
1. The energy of oxidation in the cycle is efficiently
conserved
3 NADH, 1FADH2, 1 GTP or ATP
2. Any compound that give rise to four- or five carbon
intermediates of the citric acid cycle can be oxidized by
the cycle
glutamate  a-ketoglutarate
aspartate  oxloactate
3. The citric acid cycle is amphibolic, serving in both
catabolism and an anabolism; cycle intermediates can be
drawn off and used as the starting material for variety of
biosynthetic products
Anaplerotic reactions replenish citric acid cycle intermediates
- As intermediates of the citric acid cycle are removed to serve as
biosynthetic precursors, they are replenished by anaplerotic reactions
- The most important anaplerotic reaction in mammalian liver and kideny
is the reversible carboxylation of pyruvate by CO2 to form
oxaloacetate.
Pyruvate Carboxylase
- Requires biotin
- Four identical subunits
- Biotin covalently attached
through amide linkage to the
epsilon-amino group of Lys
- First step: a carboxyl group is
attached to biotin
- Second step: the carboxyl
group is transferred to
pyruvate
- Separate active site: the long
flexible biotin arm transfer
activated carboxyl group
from the first active site to the
second active site
Long flexible arms in biochemical reaction
Pyruvate dehydrogenase complex
1. Allosteric regulation
- Inhibited by ATP, acetyl-coA, NADH, long-chain fatty acid
- Activated by AMP, CoA, and NAD+
2. Covalent protein modification
- Inhibited by phosphorylation of E1
- Kinase allosteically activated by ATP
- Thus [ATP] high  inhibit pyruvate dehydrogenase complex
Regulation of citric acid cycle
1. Three factors govern the rate of the cycle
- Substrate availability, inhibition by products, allosteric
feedback inhibition
- Substrate availability: acetyl-CoA and oxaloacetate limits
the rate of citrate formation
- Inhibition by product: citrate synthase by citrate, aketoglutarate dehydrogenase by succinyl-CoA
- allosteric feedback inhibition: ATP inhibits citrate synthase
and isocitrate dehydrogenase
2. In muscle, Ca2+, the signal for increase in demand for ATP,
acitvate isocitrate DH, a-ketoglutarate DH as well as PDH
complex
Regulation of citric acid cycle
Glyoxylate Cycle
1. Vertebrate cannot convert fatty
acid, or the acetate derived
from them, to carbohydrate
- PEP to PA and PA to AcetylCoA are so exergonic as to be
essentially irreversible
- In many organism, other than
vertebrates, the glyoxylate
cycle serve as a mechanism
for converting acetate to
carbohydrate
- Two enzymes mediates
glyoxylate cycle: isocitrate
lyase and malate synthase
- Vetebrates do not have these
enzymes
- Overall, 2 acetyl-CoA
produces1 succinate
-
-
-
-
In plant, glyoxysomes contain
all the enzymes needed for
the degradation of the fatty
acid
Acetyl-CoA from fatty acid is
converted to succinate and
then succinate is exported to
mitochondria
Succinate is converted into
malate in citric acid cycle
malate is exported to cytosol
and converted into glucose by
gluconeogenesis
Glyoxylate Cycle and citric acid cycle are coordinately regulated
-
-
-
Isocitrate dehydrogenase is
regulated by covalent
modification; phosphoylation
 inactivated  isocitrate
enter to glyoxylate cycle 
glucose
Phosphatae remove phosphate
group from isocitrate
dehydrogenase and reactive
this enzyme
Regulatory protein kinase and
phosphatase in single
polypeptide
The same intermediates of
glycolysis and the citric acid
cycle activate isocitrate
dehydrogenase wherase
inhibits isocitrate lyase