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control of metabolic reactions
•making +ve DGo‘ reactions happen
•points of control: DGo‘ and equilibrium
•multi-active enzymes: enzyme complexes and
multiple active sites
reactions with +ve DGo‘ can occur by:
• coupling with a reaction with –ve DGo‘
• –ve physiological DG due to cellular low
ratio [products]/[reactants]
1. reactions with +ve DGo‘ occur by
coupling with a reaction with -ve DGo‘
• Thus, ATP  ADP +Pi (DG<0) is coupled with
non-spontaneous reactions (DG>0)
Glucose
glucose-6-P + H20
DG = 13.8 kJ.mol-1
hexokinase
DG = -30.5 kJ.mol-1
ATP +H20
ADP +Pi
DG = -16.3 kJ.mol-1
Glucose + ATP
overall
glucose-6-P + ADP
2. Recall:
For a reaction A + B  C + D
[C] [D]
DG = DGº' + RT ln
[A] [B]
DGo' of a reaction may be positive,
and DG negative, depending on cellular
concentrations of reactants and products.
Many reactions for which DGo' is positive are spontaneous in
vivo because other reactions cause  [products] or [substrate].
any  [products] or [substrate] that moves the reaction away
from equilibrium ratio causes reaction to proceed
spontaneously forward to restore equilibrium
free energy change is related
to the equilibrium constant
(K'eq) = the ratio of
DG = DGº' + RT ln
[C] [D]
[A] [B]
 = DGº' + RT ln
[C] [D]
[A] [B]
[C] [D]
DGº' = - RTln
[A] [B]
[C] [D]
defining K'eq =
[A] [B]
DGº' = - RT ln K'eq
[products]/[reactants] at
equilibrium
At equilibrium, no
net change
so DG = 0.
I won’t be asking you to solve any
of these equations!
many reactions are near equilibrium
• then DG ~0 (no net change in free energy)
• easily reversed by changing ratio of
[products]/[substrate]
 as don’t need to overcome high DG
For A+B ↔C+D
 product A+B  C+D
 substrate A+B C+D
• enzymes that catalyse such reactions act to
restore equilibrium
• rate regulated by [products]/[reactants]
Implication:
a reaction near equilibrium may have
+ve DGo' but be spontaneous in the cell
-ve DG because other reactions
cause  [products] or [substrate].
Other reactions are FAR from equilibrium
• enzyme rate is too slow to allow products
to build to equilibrium concentration
• [substrate] builds up in excess of Keq
 DG <<<0 (highly negative)
• not affected by D[substrate] (saturated)
• essentially irreversible
• rate controlled by changing activity of
enzyme (eg allosteric interactions)
• reactions with DG <<0 are often
sites of regulation
reactions with DG <<0 are often sites
of regulation
1. Often occur early as a “committed step” in
metabolic pathways (eg AcetylCoA carboxylase)
2. most metabolic pathways are irreversible
• ≥ 1 step with -ve DG required to drive: eg PDH,
pyruvate carboxylase)
• one way street: return by a different street
3. catabolic and anabolic pathways are separate
 independent control (eg glycolysis and
gluconeogenesis (eg pyruvate carboxylase) use
different enzymes)
Pyruvate dehydrogenase
a pretty, pink multi-enzyme complex
‘gatekeeper’ to entry to citric acid cycle
http://www.brookscole.com/chemistry_d/templates/student_resources/shared_resources/animations/pdc/pdc.html
Pyruvate
dehydrogenase
2 NADH
pyruvate
dehydrogenase
2 NADH
6 NADH
regulates entry
into the citric
acid cycle of
metabolites
leaving
glycolysis
Summary
1. structure of PDH complex
3 enzymes (E1, E2, E3)
2. reactions of PDH complex
5 reactions, 5 cofactors
3. mechanism of PDH complex
lipoamide swinging arm
4. regulation of PDH complex
de/phosphorylation of E1
product inhibition of E2 and E3
Excellent animation of PDH reactions if you can access it: (not examinable, but might help understanding!)
http://www.brookscole.com/chemistry_d/templates/student_resources/shared_resources/animations/pdc/pdc.html
PDH = multi-enzyme complex
3 different ENZYMES
(non-covalently associated)
5 COFACTORS
E1: pyruvate dehydrogenase
+TPP
E2: dihydrolipoyl transacetylase + lipoamide
E3: dihydrolipoyl dehydrogenase + FAD
catalyse
5 sequential reactions
+ NADH
+CoenzymeA
overall…..
NAD+
high
energy
bond
CoA
(2C)
pyruvate
AcCoA CoA
(3C)
NADH
CO2
irreversible
multi-enzyme complex (E. coli)
• a) dihydrolipoyl transacetylase (E2)
• arranged as corners of a core cube
surrounded by an outer cube:
• b) pyruvate dehydrogenase (E1)  edges
• c) dihydrolipoyl dehydrogenase (E3)  faces
Note that
there are
many copies
of each
enzyme in
each complex
PDH structure is more complex in
other organisms
dodecahedron core=
• 12 pentagon faces
• 20 vertices (E2 trimers)
• in mammals =
+ kinase
+ phosphatase
E2 core of B. stearothermophilus
each enzyme uses a cofactor
TPP in
each E1
FAD in
each E3
2 lipoate binding domains in
each E2
pyruvate
dehydrogenase
(E1)
5 sequential reactions
In summary: 1) pyruvate is decarboxylated hydroxyethyl, requires TPP to
stabilise the intermediate. 2) hydroxyethyl oxidised to acetyl, collected by
lipoamide of E2, which gets reduced. 3) lipoamide of E2, passes acetyl to
coenzyme A  acetyl CoA. 4) lipoamide of E2, gets re-oxidised, gives its
electrons to FAD in E3 which 5) passes electrons to NAD  NADH
dihydrolipoyl
transacetylase
(E2)
dihydrolipoyl
dehydrogenase
(E3)
5
4
1
2
3
1. decarboxylation by E1
loss of CO2 conversion of pyruvate to a 2 carbon moiety
hydroxyethyl(2C)
Pyruvate
(3C)
E1 has a bound coenzyme (TPP)
that attacks pyruvate and
stabilises the intermediate
i. TPP forms a carbanion
H+ readily dissociates (due to adjacent N+)
N+ stabilises the carbanion
H+
ii. nucleophilic attack by TPP carbanion on
electron-deficient C2 of pyruvate
hydroxyethyl-TTP
CO2
releases CO2
iii. TTP stabilises the carbanion intermediate
after CO2 is lost.
can’t just remove CO2 
highly unstable intermediate
I won’t ask you
to recreate bond
rearrangements!
CO2
2. formation of acetyl by E1
+ TTP regenerated
hydroxyethyl
acetylOXIDATION
REDUCTION
- R - lys
gain of hydrogen
lipoamide
- R - lysine
E2
dihydrolipoamide
hydroxyethyl is transferred the lipoamide group of
E2, Lipoamide (= lipoic acid linked covalently to
Lysine) contains a cyclic disulfide reactive group
that can be reversibly reduced  dihydro-lipoamide
E2 uses lipoamide as a cofactor
cyclic disulphide
reversibly reduced and oxidised
lipoic acid acts as a long flexible
arm that can transfer substrates
between active sites
there are actually 2 lipoate-binding domains in
each E2.
lipoamide
= lipoic acid
covalently bound
to lysine in E2
3. trans-esterification
acetyl group transferred by E2 to CoA
= high energy
thioester bond
Thioesters: high energy bond
• Form between carboxylic acid (COOH)
and a thiol (SH) eg thiol in CoenzymeA
• eg Acetyl-CoA is common to CHO, fat and
protein metabolism
• eg.In citric acid cycle, cleavage of
thioester in succinyl-CoA provides energy
for synthesis of GTP
Lipoamide cofactor in E2
So far….
• disulfide swings to outer
shell to collect hydroxyethyl
from TPP in E1
• swings to E2 to transfer
acetyl to CoA
now we have acetyl-CoA
Remember: there are multiple copies of
 Kreb’s, FA synthesis
each enzyme in complex
next must regenerate lipoamide and
produce NADH
• So… lipoamide swings to E3 to be reoxidised
and transfer electrons to NADH via FAD
4. regeneration of lipoamide (E2) by FAD (E3)
OXIDATION
in E2
REDUCTION
in E3
5. redox
• FAD funnels electrons to NAD+ NADH
• regeneration of FAD in E3
OXIDATION
in E3
REDUCTION
in E2
NAD+
NADH + H+
ENZYME
E1: pyruvate dehydrogenase
E2: dihydrolipoyl transacetylase
E3: dihydrolipoyl dehydrogenase
COFACTOR
+TPP
+ lipoamide
+ FAD
PDH controlled by covalent
modification and product inhibition
• mammalian complex also contains kinase
and phosphatase
P
ATP
Ser
inactive E1
PDH
phosphatase
PDH
kinase
active E1
pyruvate
NAD+
AcCoA
NADH
CO2
inhibit PDH
• high energy state
P
ATP
Ser
inactive E1
PDH
phosphatase
PDH
kinase
activates
active E1
pyruvate
NAD+
AcCoA
NADH
CO2
inhibition by products
in addition to activating PDH kinase,
NADH and acetyl-CoA:
• compete with substrates for binding sites
• drive E2 and E3 in reverse (these
reactions are close to equilibrium)
• E2 not available to collect hydyrxyol from
TPP
• TPP cannot accept pyruvate
activate PDH
• low cell energy, or high available fuel
glucose
 Insulin
P
ADP
Ser
inactive E1
activates
PDH
phosphatase
PDH
kinase
active E1
pyruvate
AcCoA
NAD+
NADH
CoA
CO2
activate PDH
pyruvate overrides NADH, AcCoA
 still make AcCoA for fat when pyruvate
P
Ser
inactive E1
PDH
phosphatase
PDH
kinase
activates
active E1
pyruvate
AcCoA
NAD+
NADH
CoA
CO2
glucose
in high energy:
(high ATP, high AcCoA, high NADH)
 gluconeogenesis, fatty acid synthesis
in low energy
(low ATP, low AcCoA, )
 glycolysis
PEP
PK
pyruvate
Pyr
carbox
CO2
PDH
CO2
AcCoA
AC
Carbox
malonylCoA
FA
Synthase
fatty
acids
CO2
OAA
citric
acid
cycle
We now look at 3 other enzymes
that use ‘swinging arm’ cofactors
Pyruvate carboxylase
AcetylCoA carboxylase
Fatty acid synthase
pyruvate carboxylase
ATP
pyruvate
(3C)
+ HCO3-
ADP
oxaloacetate
(4C)
glucose
(6C)
• first reaction in gluconeogenesis
• with PEPCK to bypass pyruvate kinase
(DG<<0 in glycolysis)
• requires ATP to overcome –ve DGo‘ of
glycolysis
another good animation, if you can access it: (not examinable, but might help understanding!)
http://www.bmb.uga.edu/8010/moremen/weblinks/nucleotide/PyrCarb/PyrCarb.html
pyruvate carboxylase
•tetramer
•each monomer has 2 active sites
•uses biotin as swinging arm
in active site 1
HCO3ATP
biotin
ADP
carboxyphosphate
carboxybiotin
biotin’s
swinging
arm
Biotin carboxylation is
catalyzed at one active
site : first, ATP reacts
with HCO3(bicarbonate) to yield
carboxyphosphate.
The carboxyl from this
high energy phosphate
intermediate is
transferred to the
nucleophilic N of the
biotin ring
At active site 1:
1. bicarb + ATP
 high energy carboxyphosphate
intermediate 
2. -ve DG transfer of CO2
to biotin = carboxylation
I won’t ask you
to recreate bond
rearrangements!
HCO3ATP
biotin
ADP
carboxyphosphate
oxaloacetate
(4C)
2. biotin arm
swings to the
2nd active site,
active CO2 is transferred
from carboxybiotin to
pyruvate  OAA
pyruvate
(3C)
carboxybiotin
at active site 2:
1. CO2 leaves biotin,
2. biotin accepts a
proton from pyruvate
3. pyruvate attacks CO2
pyruvate loses a proton, becomes an enolate
nucleophile (donates e-)
I won’t ask you
to recreate bond
rearrangements!
OAA
HCO3ATP
biotin
ADP
carboxyphosphate
biotin’s
swinging
arm
carboxybiotin
oxaloacetate
(4C)
pyruvate
(3C)
Overall:
at active site 1: biotin + ATP + HCO3-  carboxybiotin + ADP + Pi
at active site 2: carboxybiotin + pyruvate  OAA + biotin
AcetylCoA carboxylase
ATP
AcetylCoA
(2C)
ADP
+ HCO3-
malonylCoA
(4C)
fatty acids
• first reaction committed step in fatty acid synthesis
•Also uses biotin as swinging
arm between two active sites
•reactions very similar to
pyruvate carboxylase
HCO3ATP
biotin
ADP
carboxyphosphate
WOW look! mechanism of carboxylation
(addition of COO-) is the same as for
pyruvate carboxylase!!! ATP-dependent
carboxylation of the biotin, carried out at
active site 1 , is followed by transfer of
the carboxyl group to acetyl-CoA at a
second active site 2 . only difference is
COO- is added to acetylCoA rather than
to pyruvate
biotin’s
swinging
arm
malony-lCoA
(3C)
Acetyl-CoA
(2C)
carboxybiotin
regulation of AcCoA-Carboxylase
The mammalian enzyme is regulated, by
 phosphorylation by cAMP dependent kinase
 inhibition when  energy (cAMP)
 allosteric control by local metabolites.
Conformational changes with regulation:
 active = multimeric filamentous complexes.
 inactive = dissociation to = monomeric form
P
fatty acid synthase
• dimer
• 6 active sites are individual domains of a large
protein
– ? developed from gene fusion
– has more catalytic activities than any enzyme!
• has two prosthetic groups thioester bonds
– thiol of cysteine (in condensing domain)
– thiol of P-pantetheine (in acyl carrier domain)
• acts as a long flexible arm transferring substrates between
active sites
has two prosthetic groups
H
H3N+
C
COO-
CH2
SH
thiol of cysteine in condensing domain
cysteine
SH
thiol of P-pantetheine
phosphopantetheine
of acyl carrier protein
CH2
CH2
-mercaptoethylamine
NH
Phosphopantetheine is covalently
linked to a serine of the acyl
carrier protein domain
C
O
CH2
CH2
pantothenate
NH
The long flexible arm of
phosphopantetheine allows its thiol
to move between active sites
C
O
HO
C
H
H3C
C
CH3 O
H2C
forms thioesters like CoA does
O
P
NH
O
O-
phosphate
CH2
CH
C
serine
residue
O
phosphopantetheine is part of CoA
SH
phosphopantetheine
of acyl carrier protein
CH2
CH2
SH
Coenzyme A
CH2
CH2
-mercaptoethylamine
-mercaptoethylamine
NH
NH
C
C
O
CH2
CH2
CH2
CH2
NH
pantothenate
HO
O
C
H
C
H2C
CH3 O
O
P
NH
O
O-
phosphate
CH2
CH
C
serine
residue
O
NH2
O
HO
C
H
H3C
C
CH3 O
H2C
H3C
pantothenate
C
NH
C
O
O
ADP-3'phosphate
P
N
N
O
O
O-
P
N
N
CH2
O
O-
O
H
H
O
H
OH
H
phosphopantetheine
-
O
P
O
O-
fatty acid synthase
2NADPH H20
2
3
2)Thioester bond between malonyl and pantetheine
3)The condensation reaction * involves decarboxylation of
the malonyl  carbanion  attacks carbonyl carbon of the
acetyl. Uses swinging arm of pantotheine
You will have done these reactions in Dr Denyer’s lectures
dimer of the multi-domain enzyme are
probably aligned in antiparallel
In the transfer step:
the growing fatty acid chain is
preferentially passed from the
pantetheine thiol of one subunit
 cysteine thiol of the other
? intra-subunit substrate
transfers also occur by
swinging arm of
pantetheine
Pant-SH HS-Cys
Cys-SH HS-Pant
Fatty Acid Synthase dimer
essential dietary cofactors:
cannot be made by mammals
• thiamine = vitamin B1 (in TPP)
– deficiency = beri-beri
– eg alcohol reduced uptakeof thiamine
 brain symptoms (brain glucose metabolism)
•
•
•
•
•
riboflavin = vitamin B2 (for FAD)
niacin = vitamin B3 (NAD)
lipoic acid
biotin
pantothenic acid (vitamin B5)
advantages of multi-active site enzymes
and multi enzyme complexes
•  diffusion distance between substrate
and active sites (usually the limiting factor in
determining the reaction rate)
 reaction rate
• chance of side reactions
– substrates stay within complex
• coordinated control of sequential reactions
Voet, Voet and Pratt (2nd Ed)
•
•
•
•
•
•
•
.DG and equilibrium pg 401
PDH pg 519 -524, regulation pg 533
TPP mechanism pg 450
thioester bonds pg 413
Pyruvate carboxylase pg 502, pg
AcetylCoA carboxylase pg 651
Fatty acid synthase pg 653 (much more detail than
you need for this lecture!)