Glycogen(n)
Glycogen(n-1)
UDP-Glucose
Glycogen(n-1)
G-1-P
UTP
PP
2P
Other tissues, like the heart, may alter this pattern.
Citric Acid Cycle (chapter 21)
- Review Fates of Pyruvate
- TCA Cycle Overview
- Source of Acetyl~SCoA
- Pyruvate Dehydrogenase Complex (PDC)
- Reactions / Structure / Regulation
Enzymes of the Citric Acid Cycle
- Reactions / Energy Summary / Amphibolic Nature
Glyoxylate Cycle (chapter 23-2)
- Glyoxysome / Mitochondrion Enzymes
Page 582
C6H12O6
C6H12O6 + 6 O2
2 C3H6O3
∆Go’ = -196 kJ/mol
6 CO2 + 6 H2O ∆Go’ = -2823 kJ/mol
Overview of the
LINKING step and
the TCA cycle.
Details will follow.
The University of Texas has played
a prominent role in the discovery of
vitamins in metabolism
University of Texas
Biochemical Institute
- vitamin discovery
Roger J. Williams
Two of the three forms of vitamin B6,
lipoic acid, avidin, folinic acid,
synthesis of vitamin B12, and pioneering
work on inositol.
(1893-1988)
William Shive
(1916-2001)
Words Coined by Roger J. Williams
Pantothenic acid, 1933
A B-vitamin. (Greek pantothen = “from everywhere”; now known to apply equally well to many other nutrients)
Williams, R. J., Lyman, C. M., Goodyear, G. H., Truesdail, J. H. and Holaday, D. Pantothenic Acid, A Growth
Determinant of Universal Biological Occurrence. J. American Chemical Society, 1933; 55:2912-27.
Folic acid, 1941
A B-vitamin. (Latin folium = leaf)
Mitchell, H. K., Snell, E. E. and Williams, R J. The Concentration of “Folic Acid.” J. American Chemical Society,
1941; 63:2284.
Avidin, 1941
A protein in raw egg white that avidly binds biotin (a B-vitamin), making it unavailable.
Eakin, R. E., Snell, E. E. and Williams, R. J. The Concentration and Assay of Avidin, the Injury-Producing Protein
in Raw Egg White. J. Biological Chemistry, 1941; 140:535-43
Pyruvate
E1(TPP)
CO2
HSCoA
E2(Lipoic Acid)
Acetyl~SCoA
Lester J. Reed
(UT 1948-1997)
Page 770
Eli Lilly Award - 1958
Merck Award - 1994
Figure 21-6
The five reactions of the PDC
NAD+
E3(FAD)
NADH
Mitochondria: Site of the linking step and the TCA cycle
This organelle has an oxidizing
environment, and interesting
evolutionary history
The “linking step”: PDC
Be sure to take your vitamins: Five cofactors are used in the PDC
Page 769
Figure 21-3a: Electron micrographs of the E. coli pyruvate dehydrogenase
multienzyme complex. (a) The intact complex, (b) dihydrolipoyl
transacetylase (E2) “core”.
Five Reactions of the PDC
Overall linking step reaction is misleadingly simple:
Pyr + NAD+ + CoA Æ AcCoA + NADH + CO2
Æ
E1 uses a TPP cofactor. In PDC the hydroxyethyl TPP is
not released as an aldehyde, as in pyr decarboxylase, but
passed to lipoic acid on E2.
Page 605
Figure 17-27 Reaction mechanism of pyruvate
decarboxylase.
Figure 21-8 Domain structure of the dihydrolipoyl
transacetylase (E2) subunit of the PDC.
Page 773
E2
E2 is the PDC core enzyme; it
spontaneously assembles. In
bacteria it forms a trimer and sits on
the 3-fold of the aggregate structure.
Figure 21-12a: X-Ray structure of E1 from P. putida
branched-chain a-keto acid dehydrogenase. A surface
diagram of the active site region shows TPP in a deep
cleft that can be reached by the mobile E2 arm system.
Page 776
E1
TPP
Lipoyllysine arm is very mobile
Ac-lipoamide reaches active site with CoA
Reduced lipoic acid is re-oxidized by E3
Figure 21-13a: X-Ray structure of dihydrolipoamide
dehydrogenase (E3) from P. putida in complex with FAD
and NAD+ shows physical arrangement of redox pair.
Page 777
E3
Figure 21-14
Catalytic reaction
cycle of
dihydrolipoyl
dehydrogenase.
Page 778
E3
Page 780
Figure 21-16 The reaction transferring an electron pair from
dihydrolipoyl dehydrogenase’s redox-active disulfide in its
reduced form to the enzyme’s bound flavin ring.
E3
Mammalian Complex:
60 E2 (52 kDa)
30 E1(α2β2 154 kDa)
12 E3 dimers (110 kDa)
+ ~6 binding proteins
+ ~3 kinase (~62 kDa)
Page 774
+ ~3 phosphatase (~100kDa)
Figure 21-11c: Electron microscopy–based images of the bovine kidney
pyruvate dehydrogenase complex at ~35 Å resolution. (c) A cutaway
diagram as in Part b but with E3 dimers (Fig. 21-13a) shown at 20 Å
resolution (red) modeled into the pentagonal openings of the E2 core.
PDC is regulated by products
High concentrations of NADH and/or AcCoA
can run reactions 3 & 5 backward
Page 781
Figure 21-17b
Factors controlling the activity of the PDC.
(b) Covalent modification in the eukaryotic complex.
On to the TCA cycle
Hans Krebs
1937
C2
C4
C6
C4
Page 766
C6
C4
C5
Simplified
TCA Cycle
Page 782
TCA Cycle Enzymes
Figure 21-18a: Conformational changes in citrate
synthase. (a) Space-filling drawing showing citrate
synthase in the open conformation. (b) closed, substratebinding conformation.
Figure 21-19 : Mechanism and stereochemistry of the
citrate synthase reaction.
Aha! Enolate anion as a
nucleophile.
Aconitase removes,
and then adds
back, water
Aconitase has a 4 Fe-4S cluster.
The FeS cluster carries out NO
redox function, but interacts
directly with an organic
substrate.
In humans, a CYTOSOLIC form
doubles as an iron monitor,
regulating transcription from
iron response elements (IRE).
Page 784
Mechanism and stereochemistry of the aconitase reaction.
Page 785
Figure 21-21 Probable reaction
mechanism of isocitrate
dehydrogenase.
Oxidation creates a carbonyl electron sink to facilitate βdecarboxylation
The five reactions of the KGDC are similar to those of PDC,
and the structure of KGDC is similar to the 24-mer PDC.
Page 787
succinyl-CoA synthetase: the only direct
phophorylation in TCA - step 1
Figure 21-22a: Reactions catalyzed by succinyl-CoA
synthetase. Formation of succinyl phosphate, a
“high-energy” mixed anhydride.
Page 787
succinyl-CoA synthetase - step 2
Figure 21-22b: Reactions catalyzed by succinyl-CoA
synthetase. Formation of phosphoryl–His, a “highenergy” intermediate.
Succinate dehydrogenase
- membrane bound enzyme
FADH2
Page 787
FAD
Figure 21-23 Covalent attachment of FAD to a His
residue of succinate dehydrogenase.
Page 790
Standard Free Energy Changes (∆G°’) and Physiological
Free Energy Changes (∆G) of TCA Cycle Reactions.
Simplified
TCA Cycle
Regulation of the
citric acid cycle.
Cit Synth
ICDH
Page 791
Flux through the
system is largely
controlled by
concentrations of
reactants and
PRODUCTS, esp
NADH, at
irreversible steps.
PDC
KGDC
Another
representation
of TCA
regulation
, NADH
Page 793
Amphibolic
functions of the
citric acid cycle.
The intermediates
of the TCA cycle
are chemically very
useful and are
drawn off for a
variety of tasks.
Without
CARRIERS, the
cycle slows
Anaplerotic Reactions:
1) Pyruvate Carboxylase (has requirement for AcCoA
– that makes sense!)
pyr + CO2
+ ATP
OAA + ADP
2) Malic E
pyr + CO2 + NADPH
3) Transamination Reactions:
Ala
Pyr
Asp
OAA
Glu
KG
L-Mal + NADP+
Page 851
Figure 23-10:The glyoxylate cycle. Why plants can
convert lipid to sugar and you can’t.
The Glyoxylate
shunt, found in
plants and some
microbes,
allows synthesis
of glucose from
lipid derived
AcCoA. Two
novel enzymes
short work with
TCA to create
the shunt.
Radioisotopes greatly facilitate
metabolic mapping.
Putting label at
differing places
allows
researchers to
follow enzyme
activities and
construct
pathways.
14C
14C
Dating
is generated from N2 in atmosphere at ~constant rate.
Living systems take it up, until they die. From then on,
normal fraction of 14C decays with half live ~5700 years.