TCA (Krebs) Cycle
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Transcript TCA (Krebs) Cycle
The Tricarboxylic Acid Cycle
O
H 3C
C
(2 O2)
2 CO2 + CoA + 12 e– + 2 H2O
SCoA
Lecture 23-24
Baynes & Dominiczak, Chapter 13
Gene C. Lavers, Ph.D.
[email protected]
home.nyu.edu/~gcl1
©Copyright 1999-2004 by Gene C. Lavers
No part of this presentation may be reproduced by any mechanical, photographic, or electronic process, or in
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Amphibolic Roles
Energy production vs. Biosynthesis
Krebs TCA Cycle
Mitochondrion
Tricarboxylic acid cycle (TCA), Krebs cycle,
or citric acid cycle: located in mitochondrion;
common 8-Rx oxidative pathway for all fuels.
Two major metabolic roles: energy
production and biosynthesis.
4 oxidative steps: capture high DG e– in 3
NADH and 1 FADH2; transfer to ETS for ATP.
Substrate level phosphorylation: 1 GTP.
8 reactions: consumes acetate as acetyl CoA;
releases 2 CO2; yields GTP, e–,
acetyl CoA: co-substrate from carbohydrates,
fat, and protein, condenses with OAA, forms
citrate, then 7-Rx OAA.
NADH and FADH2: capture and transfer high
DG e– to ETS.
Intermediates: enter and leave cycle for
oxidation or biosynthesis, respectively.
No oxygen used by TCA.
No ATP produced directly in TCA: ETS.
Fig. 13.1 Amphibolic nature of ©Copyright 1999-2004 by Gene
TCA cycle.
C. Lavers
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Acetyl CoA
Common catabolite
TCA Cycle
Mitochondrion
Glucose, fructose, galactose, mannose: yield
Fig 13.2 Metabolic sources
of acetyl CoA.
pyruvate (pyr).
Lipid fatty acids: yield acetyl CoA
Protein amino acids are:
Glucogenic: ala cys gly ser thr trp pyr glc
TCA: a-KG succ-CoA fumarate OAA glc
arg glu gln his pro a-KG
ile met thr val succinyl CoA
asp phe tyr fumarate
asn asp OAA
Ketogenic: ile, leu, trp acetyl CoA
leu, lys, trp, phe, tyr acetoacetyl CoA Acetyl CoA
Pyruvate acetyl CoA + CO2
(oxidative decarboxylation)
3-C
2-C
1-C
Acetyl CoA: substrate for TCA, fatty acid synthesis &
cholesterol synthesis, protein N-terminal acetylations …
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C. Lavers
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2 Pyruvate Acetyl CoA + Oxaloacetate
2 (C-C-C) 1 C-C + C-C-C-C
TCA Cycle
Mitochondrion
PC balances OAA synthesis for acetyl CoA oxidation*
Fig 13.3 Source of OAA and
acetyl CoA.
Pyruvate: common intermediate for both cosubstrates of TCA cycle.
Oxidative decarboxylation: pyruvate to
acetyl CoA via PDH
Carboxylation: pyruvate to OAA via PC,
ATP required to make C—C bond.
Catalytic amounts of OAA: replenished by
TCA cycle: thus, can oxidize large [acetyl CoA].
Citrate synthase: aldol condensation yields
6-C product, citric acid (citrate).
Stereo specific, asymmetric center (chiral).
_____________________________________
* When blood [glucose] low, PC increases [OAA] for
gluconeogenesis (during lipolysis of fats for ATP)
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C. Lavers
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Pyruvate Carboxylase
TCA Cycle
ATP ADP + P
Mitochondrion
Pyruvate: converted to/from many
compounds: lactate, ala, OAA.
Routed to:
Gluconeogenesis
Fatty acid synthesis
TCA oxidation
PC fixes CO2:
Fig 13.4 Role of covalently-linked
biotin in OAA synthesis
uses biotin to form amide
bond; most amide bonds formed require
energy from ATP hydrolysis.
Biotin: vitamin 1-carbon carrier
covalently bonded to PC via an amide
bond.
PC: an anaplerotic enzyme.
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Pyruvate Dehydrogenase (PDH)
TCA Cycle
Mitochondrion
Multienzyme complex
3-enzymes
use
E1 PDH
E2 DHLT
TPP
lipoamide
CoA-SH
E3 DHLD
Pyruvate acetyl CoA
NAD+ NADH + H+
FAD FADH2 .
Lipoamide: swinging arm
Fig. 13.5 Mechanism of PDH.
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PDH Reactions
Lipoic Acid 2-carbon carrier, transthioesterification
TCA Cycle
5-Coenzymes
Thiamine pyrophosphate: attacks
keto-carbon-2 of pyruvate, CO2
released.
Lipoamide: ‘swinging arm’ forms
thioester with acetate group.
Transthioesterification: CoA-SH
replaces lipoamide reduced form
5-membered ring:
R-S—S-R vs. R–SH + Acetyl-S–R
Oxidation to R-S—S-R: uses FAD
Oxidation of FADH2: resets to FAD
PDH re-initialized for next
pyruvate.
Fig. 13.5 Lipoic acid of PDH.
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TCA Cycle Reactions
8 Reactions
Krebs TCA Cycle
Pathway Outline
1. Aldol condensation: citrate.
2. Isomerization: isocitrate.
3. Oxidative decarboxylation:
a-KG + CO2.
4. Oxidative decarboxylation:
succinyl CoA + CO2.
5. Substrate level
phosphorylation: succinate +
GTP.
6. Dehydrogenation: fumarate.
7. Hydration: malate.
8. Dehydrogenation: oxaloacetate
(OAA).
Fig. 13.7 TCA cycle.
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Stereospecificity of Enzyme Reactions
Aconitase creates chirality from achiral citrate
TCA Cycle
3-Point Contact
Citrate: achiral, meso stereoisomer.
Aconitase binds: 3-groups from OAA moiety
of citrate.
–OH 1,2-shift: from central C3 to C2, via
dehydration/hydration on OAA moiety.
Isocitrate C2: chiral center, optically active,
rotates polarized light. Also C3
CH2COO–
Fig. 13.8 Stereochemistry of the
aconitase reaction.
HO C—COO–
CH2COO–
citrate
At equilibrium: 90%
C
COO–
CH2COO–
H–C—COO–
–
C*—COO
HO
H
COO–
H
[cis-aconitate] isocitrate
3%
7%
Bound to enzyme
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C
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Stereospecificity of NAD+ reduction
TCA Cycle
Deuterium isotope (heavy hydrogen)
Hydride transfer, H:–
Each NAD
dehydrogenase add
H:– from only one side
of the pyridine ring.
Stereo addition
Fig. 13.9 Stereochemistry of reduction of NAD+.
D
H
3
H
D
H
3
CONH2
D
H
3
3
N
N1
N
N
R
R
R
R
©Copyright 1999-2004 by Gene
C. Lavers
illustrated by G3PDH
and AD (alcohol
dehydrogenase).
Chemists used heavy
hydrogen (D) as
unequivocal markers
of stereoselectivity.
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ATP yield per Glucose
Glycolysis + TCA Cycle
Energy yield
8 + 6 + 24 = 38 ATP
–2 ATP, + [2 (2 ATP) + NADH 3 ATP] = – 2 + [10] = 8 ATP
2 (1 NADH 3 ATP) = 6 ATP
2 (3 NADH 9 ATP; 1 FADH2 2 ATP; 1 succinyl CoA 1 GTP: 12) = 24 ATP.
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From Energy:
686 kcal/mole Glc
7.3 kcal /ATP
Calc. 94 ATP yield.
From Biochemistry:
Calc 38 (36*) ATP
Found: 36 ATP
Efficiency= 36/94
= 38 %
Internal
combustion engine:
~ 40% efficient.
6
24
Fig. 13.10 ATP yield from glucose during oxidative metabolism.
*Glycerol shuttle
2 ATP/NADH
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Regulation of the TCA cycle
Several levels
TCA Cycle
Mitochondrion
Availability of NAD+: a series of dependencies:
NADH consumption for oxidative phosphorylation.
Rate of ATP utilization.
Mitochondrial NAD+ provides link between work (ATP utilization)
and fuel consumption.
OAA: catalytic amounts
Regulatory enzymes:
pyruvate dehydrogenase: supply of acetyl CoA regulated by several
allosteric and covalent modifications.
PDH kinase: NADH and acetyl CoA inactivate.
PDH phosphatase: pyruvate, CoA, NAD+ activate.
Isocitrate dehydrogenase: allosteric inhibited by ATP and NADH.
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PDH Complex Reactions
TCA Cycle
Introduction
Pyruvate acetyl CoA
a-ketoacid dehydrogenases for pyruvate, glutarate (leu,ile,val)
High
ADP, NAD+
CoA
ATP, NADH
acetyl CoA
Products
Products
Low
PDH K Ph
ATP, NADH a a i
acetyl CoA
a a i
ADP, NAD+ i
i a
CoA
i
i a
Pyruvate flux:
regulated by PDH and
NAD+ availability; product inhibited by
NADH & acetyl CoA (i, i, a).
PDH activity, vit deficiency:
thiamine(B1, beriberi, muscle weakness &
neurologic disease), riboflavin (B2), niacin
(B3, pellagra) panthothenate for CoA.
Fig. 13.11 Regulation of PDH. TA=trans-
Acetylase(E2); DHLD = DiHydLipoic
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Dehydrog(E3)
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Regulation of the TCA cycle
PDH inhibited
TCA Cycle
Fasting-Starved State
During fasting or starvation blood glucose is maintained:
NADH &
acetyl CoA are high (from FA oxidation, earlier lecture), PDH inhibited:
promotes gluconeogenesis.
High NADH: ADP ATP high (in ETS), phosphorylation of key enz-P
High NADH: 13BPG G3P (gluconeogenesis) glucose
Ala, Asp, lactate pyruvate OAA 13BPG glucose
Other Regulatory enzymes: phosphorylated (glucagon influence)
TCA
Isocitrate dehydrogenase: allosteric inhibited by ATP and NADH.
Glycolysis
pyruvate kinase: inhibits pyruvate formation from PEP.
F26BPase: removes F26BP stimulation of glycolysis (F26BP F6P).
Glycogen
Glycogen phosphorylase: blocks final depletion, ready for synthesis.
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C. Lavers
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The Tricarboxylic Acid Cycle
END
©Copyright 1999-2004 by Gene
C. Lavers
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