Download Part 5 Coenzyme-Dependent Enzyme Mechansims

King Saud University
College of Science
Department of
Biochemistry
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Part 5
Coenzyme-Dependent Enzyme
Mechanisms
Professor A. S. Alhomida
1
2
Thiamin
3
Thiamin
Pyrimidine
Thiazole
Thiamine contains two heterocyclic rings, primidine and thiazole
participate in the formation of carbanion-TPP
4
Conversion of Thiamin into Coenzyme
Form (TPP)
CH3
CH3
N
N
N
H2C
NH2
N
TPP synthetase
H2C
H
N
N
ATP
S
AMP
NH2
H
S
O
H3C
CH2CH2OH
H3C
CH2CH2O
O
P O P
O
Thiamin
O
Thiamin pyrophosphate (TPP)
5
O
Structure of TPP, Cont’d
6
7
Wet Beri-Beri
8
9
Decarboxylatoion Reactions
10
Decarboxylatoion Reactions
• Decarboxlation of carboxylic acid leads to the
formation of CO2 and a carbanion
• CO2 is a stable molecule, whereas the
carbaion is a high-energy molecule that
cannot exit for long under the biochemical
conditions
• The main barrier to decarboxylation is the
formation of the carbanion
• The decarboxylation will be facilitated when a
mechanism exists to stabilize the carbanion
produced by decarboxylation
11
Decarboxylation Reaction, Cont’d
R1
R2
O
C
C
R3
Carboxylic acid
O
R2
R1
C
C
+
O
R3
O
CO2
(stable)
Carbanion
(unstable)
12
Decarboxylatoion Reactions, Cont’d
• How can this be accomplished?
• If carbanion is adjacent to an electrondeficient group such as the carbonly group in
a ketone, ester, aldehyde or carboxlyic acid
• It will be stabilized by delocalization of the
electron pair
13
Decarboxylatoion Reactions, Cont’d
• b-Keto acids readily undego decarboxylation,
whereas the carboxylic acid that have no
carbnoly group in the b-position are stable to
decarboxylation under physiological
conditions
• Molecules such as acetic acid, or butyric acid
undergo decarboxylation only under extreme
conditions such as fusion with solid NaOH
14
Decarboxylatoion Reactions, Cont’d
Electron sink
O
H3 C
C
H
C
O
O
H3 C
C
H
b-Ketoacid
O
C
O
H
C
H3 C
C
H
C
+
C
H
H
Carbanion
O
O
Enolate ion
Carbanion stabilization by
delocalization of the
electron pair
15
Decarboxylatoion Reactions, Cont’d
No Electron sink
H3 C
H
H
C
C
H
H
O
O
H3 C
C
O
C
O
H
C
+
C
H
O
Not b-ketoacid
16
Decarboxylatoion Reactions, Cont’d
• How can a decarboxylation reaction be
catalyzed?
• Decarboxylation of a b-keto acid entails the
formation of an enolate ion that is still quite
unstable in neutral pH
• Any interaction with an enzyme that stabilizes
the negative charge will be helpful in the
catalyzing decarboxylation
17
Decarboxylatoion Reactions, Cont’d
• An enzyme-bound enolate can be stabilized by
a positive charged entity such as the proton of
an acidic group or the positive charge of metal
ion placed near the carbonly oxygen
• Stabilization of the enolate lowers the
activation energy for the reaction and
increases the rate
18
Stabilization of Enolate at Active Site
by Acid
General acid donates
hydrogen bond to the bcarbonly group of a bketo acid
General acid donates a H+ to
the enolate anion resulting an
enol intermediate
B
B
H
O
H3 C
C
H
H
C
H
b-Keto acid
O
O
O
H
H3 C
C
O
C
C
C
H
O
Enol intermediate
19
Stabilization of Enolate at Active Site
by Metal Ion
Metal ion polarizes
hydrogen the b-carbonly
group of a b-keto acid
via coordination bond
Metal ion stabilizes the enolate
anion via an electrostatic bond
M2+
O
H3 C
C
M2+
H
C
H
b-Keto acid
O
O
O
H
H3 C
C
O
C
C
C
H
O
Enolate intermediate
20
Decarboxylatoion Reactions, Cont’d
• The enol intermediate is much more stable
than the enolate and it is the intermediate in
enzymatic reaction rather than the enolate
• Conversion of the b-carbonly group into a
protonated imine also facilitates the
decarboxylation
• The pH of an imine is near 7, so that under
biochemical conditions the imine-nitrogen can
be positively charged and acts as a very
effective electron sink
21
Stabilization of Imine
Protonated nitrogen of
imine
R1
H3 C
R1
H
N
C
H
C
H
Electron sink
H
N
O
H3 C
C
O
b-iminium ion carboxylic
acid
C
Dipolar
R1
O
N
H
C
H
H3 C
H
C
C
H
H
Carbanion imine
Enamine
22
+
C
O
Decarboxylatoion Reactions, Cont’d
• Decaboxylation of protonated imine, (bcationic imine,b-iminium ion) leads to the
formation of an enamine
• Enamine is a lower-energy intermediate than
an enolate
• b-Iminium ion nitrogen carries full positive
charge comparing with b-carbonyl group
(partially positive charge)
• b-Iminium ion facilitates decarboxylation
even more effectively than does a b-carbonly
group
23
Decarboxylatoion Reactions, Cont’d
• b-Iminium ion facilitates decarboxylation
even more effectively than does a b-carbonly
group
• If the keto group of a b-ketoacid is converted
into a protonated imine, the rate of
decarboxylation will be greatly enhanced
• As example of enzymatic decarboxylation via
forming imine intermediate:
– Acetoacetate decarboxylase
24
Decarboxylatoion Reactions, Cont’d
• The enzymes catalyze the
dehydrogenations and decarboxylations of
b-hydroxy acids do NOT form imines before
decarboxylation
• They require a divalent cation to facilitate the
decarboxylation through coordination with the
b-carbonly group via providing positive
charge to help stabilize the carbanion
intermediate resulting from decarboxylation
25
Decarboxylatoion Reactions, Cont’d
• Example of enzymes catalyze b-hydroxy
acids:
– Malic enzyme
– Isocitrate dehydrogenase
– 6-phosphogluconate dehydrogenase
26
Decarboxylation of a-Keto Acid
27
Decarboxylation of a-keto Acid
• The decarboxylation of a-keto acids occurs
frequently in biological systems
• It is not obvious that a-keto acids should
decarboxlyate readily, because
decaroxylation of these acids would NOT
produce a stabilized carbanion
• These acids undergo a chemical modification
before decarboxylation, which converts them
into structures resembling b-keto acids
28
Decarboxylation of a-keto Acid, Cont’d
• This chemical modification is facilitated by
TPP
• How does TPP function in decarboxylation of
a-keto acids?
• TPP can undergo a variety of chemical
reactions
• It contains a thiazolium ring can easily be
deprotonated and forms a Zwitter-ion which
reacts as a nucleophile through the carbanion
intermediate
29
Comparison Studies
R` N
2
H
H
H
S
CH3
Thiazolium
R` N
R
CH3
2
O
R` N
R
Oxazolium
2
N
H
R
CH3
Imidazolium
30
Comparison Studies, Cont’d
• C-2 oxazolium is more acidic and the oxygen
has no d orbitals, however, it is not catalyst
• Because C-2 is too stable to add weak
electrophilies and unreactive at neutral pH
• C-2 imidazolium is very slow to generate
carbanion intermediate
• Both oxazolium and imidazolium ions are
thermodynamic stable at pH 7
31
Comparison Studies, Cont’d
• The are NOT suitable for conezyme function
as thiazolium ion
• The thiazolium ion is the only cone of the
three that Is suitable on thermodynamic and
kinetic grounds
32
Biochemical Reactions of TPP
• TPP is a coenzyme for two types of reactions:
• (1) Decarboxylation
– (1) Nonoxidative decarboxylation
• Yeast pyruvate decarboxylase
– (2) Oxidative decarboxylation
• a-keto acid dehydrogenases
• (2) Transketolaction
– Transketolases
33
TPP-Dependent Enzymes
O
O
O
TPP, RCHO
TPP
R
COO
H
a-Keto acid
Acetaldehyde
TPP,
FAD,
O2
O
O
Acetic acid
TPP,
lipoamide,
CoASH,
NADH, FAD
OH
a-Hydroxyacetyl
O
SCo A
Acetyl-CoA
34
Mechanism of Pyruvate
Dehydrogenase (PDH)
Complex
35
Reaction of PDH Complex, Cont’d
36
Structure of PDH Complex
• The transacetylase core
(E2) is shown in red, the
pyruvate
dehydrogenase (E1) in
yellow, and the
dihydrolipoyl
dehydrogenase (E3) in
green
37
Structure of Transacelylase
• Each red ball
represents a trimer of
three E2 subunits
• Each subunit consists
of three domains:
(1) lipoamide-binding domain
(2) Small domain for
interaction with E3
(3) Large transacetylase
catalytic domain
• All three subunits of the
transacetylase are
shown in red
38
Structure of PDH Complex
• The PDH complex is comprised of multiple
copies of three separate enzymes:
E1: Pyruvate dehydrogenase (or decarboxylase) (2030 copies)
E2: Dihydrolipoyl transacetylase (60 copies)
E3: Dihydrolipoyl dehydrogenase (6 copies)
39
Structure of PDH Complex, Cont’d
40
Structure of PDH Complex, Cont’d
• The complex also requires 5 different
coenzymes:
(1) TPP
(2) CoA
(3) NAD+
(4) FAD+
(5) Lipoamide
• TPP, lipoamide and FAD+ are tightly bound to
enzymes of the complex whereas the CoA
and NAD+ are employed as carriers of the
products of PDH complex activity
41
The coenzymes and Prosthetic
Groups of PDH Complex
Coenzyme
Location
Function
TPP
Bound to E1
Decarboxylates Pyr,
yielding HE-TPP
carbanion
Lipoate
Covalently linked to
Lys on E2 (lipoamide)
Accepts HE carbanion
from TPP as an acetyl
group
CoA
Coenzyme for E2
Accepts the acetyl
group from acetyldihdrolipoamide
FAD
Bound to E3
Reduced by
dihdrolipoamide
NAD+
Coenzyme for E3
Reduced by FADH2
42
Structure of PDH Complex, Cont’d
• PDH complex is a noncovalent assembly of
three different enzymes operating in concert
to catalyze successive steps in the
conversion of pyruvate to acetyl-CoA
• The active sites of all three enzymes are not
far removed from one another, and the
product of the first enzyme is passed directly
to the second enzyme and so on, without
diffusion of substrates and products through
the solution
43
Lipoic acid
• Lipoic acid is a coenzyme found in PDH
complex and a-KGDH complex, two
multienzymes involved in a-keto acid
oxidation
• Lipoic acid functions to:
– Couple acyl group transfer
– Electron transfer during oxidation and
decarboxylation of a-ketoacids
• No evidence exists of a dietary lipoic acid
requirement in humans; therefore it is not
considered a vitamin
44
Structure of Lipoamide
S
• Lipoamide includes a
dithiol that undergoes
oxidation/ reduction
• It acts as a carrier and
an redox agent
CH2
CH2
S
lipoic acid
CH
O
CH2 CH2 CH2 CH2 C
NH
lysine
NH (CH2)4 CH
lipoamide
C
O
2e + 2H+
HS
CH2
HS
CH
CH2
NH
O
CH2 CH2 CH2 CH2 C
NH (CH2)4 CH
dihydrolipoamide
C
45
O
Structure of Lipoamide, Cont’d
1. The carboxyl at the
end of lipoic acid's
hydrocarbon chain
forms an amide bond
to the side-chain
amino group of a
lysine residue of E2
yielding lipoamide
S
CH2
CH2
S
CH
lipoic acid
O
CH2 CH2 CH2 CH2 C
NH
lysine
NH (CH2)4 CH
lipoamide
C
O
2e + 2H+
HS
CH2
CH2
HS
CH
NH
O
CH2 CH2 CH2 CH2 C
NH (CH2)4 CH
C
46
O
Structure of Lipoamide, Cont’d
2. A long flexible arm,
including hydrocarbon
chains of lipoate and
the lysine R-group,
links each lipoamide
dithiol group to one of
2 lipoate-binding
domains of each E2
S
CH2
CH2
S
CH
lipoic acid
O
CH2 CH2 CH2 CH2 C
NH
lysine
NH (CH2)4 CH
lipoamide
C
O
2e + 2H+
HS
CH2
CH2
HS
CH
NH
O
CH2 CH2 CH2 CH2 C
NH (CH2)4 CH
C
47
O
Structure of Lipoamide, Cont’d
3. Lipoate-binding
domains are
themselves part of a
flexible strand of E2
that extends out from
the core of the
complex
S
CH2
CH2
S
CH
lipoic acid
O
CH2 CH2 CH2 CH2 C
NH
lysine
NH (CH2)4 CH
lipoamide
C
O
2e + 2H+
HS
CH2
CH2
HS
CH
NH
O
CH2 CH2 CH2 CH2 C
NH (CH2)4 CH
C
48
O
Structure of Lipoamide, Cont’d
4. The long flexible
attachment allows
lipoamide functional
groups to swing
between E2 active
sites in the core of the
complex and active
sites of E1 and E3 in
the outer shell
S
CH2
CH2
S
CH
lipoic acid
O
CH2 CH2 CH2 CH2 C
NH
lysine
NH (CH2)4 CH
lipoamide
C
O
2e + 2H+
HS
CH2
HS
CH
CH2
NH
O
CH2 CH2 CH2 CH2 C
NH (CH2)4 CH
C
49
O
Structure of Lipoamide, Cont’d
5. E3 binding protein
that binds E3 to E2
also has attached
lipoamide that can
exchange of reducing
equivalents with
lipoamide on E2
S
CH2
CH2
S
CH
lipoic acid
O
CH2 CH2 CH2 CH2 C
NH
lysine
NH (CH2)4 CH
lipoamide
C
O
2e + 2H+
HS
CH2
HS
CH
CH2
NH
O
CH2 CH2 CH2 CH2 C
NH (CH2)4 CH
C
50
O
Structure of Lipoamide, Cont’d
6. Organic arsenicals
are potent inhibitors of
lipoamide-containing
enzymes such as
Pyruvate
Dehydrogenase
H2O
HS
R'
As O
S
R'
+
As
HS
S
R
R
7. These highly toxic
compounds react with
“vicinal” dithiols such
as the functional group
of lipoamide
51
Formation of TPP-carbanion
(Active Form)
52
Formation of TPP-carbanion
H
N
H
H
B:
CH2
H3C
N
CH3
BH+ O
C
Glu
N
N
H
H
S
CH2
R
H3C
B:
N
N
CH3
H
O
C
O
O
Glu
53
S
R
Formation of TPP-carbanion, Cont’d
Electron sink to stabilize the
negative charge
H
N
H
CH2
H3C
N
N
CH3
S
R
O
C
O
Glu
54
Mechanism of PDH Complex
55
Mechanism of PDH Complex
CH3
R1
N
S
R2
Pyruvate
decarboxylase
TPP carbanion
C
CH3
C
O
C
O
BH
O
Pyruvate
56
Decarboxylation step
CH3
R1
N
R1
CH3
N
C
C
OH
Enz
C
O
S
R2
CH3
CH3
O
Tetrahedral intermediate
C
C
OH
Enz
C
O
S
R2
O
Transition state
57
Delocalization of electrons into
iminium electron sink
R1
CH3
N
..
S
CO2
R2
CH3
C
C
CH3
S
R2
Electrophile
R1
CH3
N
OH
Enz
Nucleophile
Dipolar
C
OH
C
Enz
Carbanion of HETPP
Resonance form of hydroxyethyl-TPP
58
S
S
Enz
Electron sink to stabilize
the negative charge
CH3
Dihydrolipoamide
R1
CH3
N
C
S
R2
C
OH
Enz
Hydroxyethyl-TPP
S
S
BH
Enz
Oxidized (dihydrolipoamide(
59
B:
Oxidation and
transferring step
R1
CH3
CH3
N
S
H
C C O
R2
Enz
S
SH
Enz
Tetrahedral intermediate
CoA SH
Dihydrolipoyl
transacetylase
CH3
N
S
CH3
CoA-S
H
R1
R2
C
Enz
C O
B:
S
BH+
SH
TPP
Enz
Acetyl-dihyrolipoamide (Thioester)
60
Oxidation step
HS
SH
Enz
Reduced (dihyrolipoamide)
FAD
CH3
C
NADH + H+
Dihydrolipoyl DH
O
SCoA
Acetyl-CoA
FADH2
S
NAD+
S
Enz
Oxidized (dihydrolipoamide(
61
Structure of Dihydrolipoly
Transacelyase
• Domain structure of the
dihydrolipoyl
transacetylase (E2)
subunit of the PDH
complex
62
Structure of Dihydrolipoly
Transacelyase, Cont’d
• X-Ray structure of a
trimer of A. vinelandii
dihydrolipoyl
transacetylase (E2)
catalytic domains
63
Structure of Branched-chain aKeto Acid DH Complex
• X-Ray structure of E1
(PDH) from P. putida
branched-chain a-keto
acid dehydrogenase
• The a2b2
heterotetrameric protein
• The TPP binds at the
interface between a and
b subunits
64
Structure of Branched-chain a-Keto
Acid DH Complex, Cont’d
• X-Ray structure of E1
(PDH) from P. putida
branched-chain a-keto
acid dehydrogenase
• A surface diagram of the
active site region
• The lipoyl-lysyl armof the
E2 lipoyl domain has
been model into channel
• The TPP-substrate
adduct in an enamineTPP form
65
Structure of Dihdrolipoamide DH
• X-Ray structure of
dihydrolipoamide
dehydrogenase (E3) from P.
putida in complex with FAD
and NAD+
• The homodimeric enzyme
• One subunit is gray and the
other is colored according to
the domain with its FADbinding domain
66
Structure of Dihdrolipoamide DH,
Cont’d
• X-Ray structure of
dihydrolipoamide
dehydrogenase (E3) from
P. putida in complex with
FAD and NAD+
• The active site of the
enzyme region
• The redox-active portions
of the bound NAD+ and
FAD is shown
67
Mechanism of Dihydrolipoyl DH
• Catalytic reaction cycle
of dihydrolipoyl
dehydrogenase
• It is similar to the
catalytic reaction cycle
of glutathione reductase
• However, glutathione
reductase uses NADPH
instead of NAD+
68
Catabolism of Branched-Chain
Amino Acid
O
O
SCoA
O
O
O
NH3
O
Isoleucine
a-Ketoacid DH
Complex
O
O
SCoA
O
O
O
NH3
CoA
CO2
O
Leucine
O
O
NH3
Valine
SCoA
O
O
O
O
69
Transketolase
70
Reaction of Transketolase
CH2OH
H
CH2OH
C
O
C
H
C
OH
transketolase
HO
H
C
C
H
OH
CH2-OPO 3H2
D-xylulose-5-phosphate
C
O
HO
C
H
H
C
OH
H
C
OH
CH2-OPO 3H2
H
C
OH
3-phosphoglyceraldehyde
O
H
C
OH
H
C
OH
TPP
CH2-OPO 3H2
D-ribose-5-phosphate
H
O
C
+
H
CH2-OPO 3H2
septulose-7-phosphate
71
C
OH
Structure of Transketolase
3- D Structure of yeast
72
Structure of Transketolase
• Baker's yeast
(Saccharomyces
cerevisiae)
• The coloring scheme
highlights the 2nd structure
and reveals that
transketolase is a dimer
• TPP has been substituted
by 2,3'-deazo-thiamin
diphosphate which is
shown
• Ca2+ (blue-gray) can be
seen complexed with the
diphosphates
73
• Transketolase is a homodimeric
enzyme containing two molecules of
noncovalently bound thiamine
pyrophosphate
74
Mechanism of Transketolase
75
Mechanism of Transketolase
CH3
B:
R1
N
S
R2
R1
CH3
C
Enz
N
H
S
1
R2
C
CH2OH
C
O
HO
C
H
H
C
OH
BH
CH2O P
76
Xylulose-5-phosphate
R1
CH3
CH2OH
N
S
R2
H
B:
C
C
OH
O
C
H
H
C
OH
R1
CH3
N
..
S
CH2OH
C
OH
C
R2
CH2O P
Ribose-5-phosphate
O
H
C
R1
CH3
H
CH2OH
C
OH
N
Dihydroxyethyl-TPP
S
C
C
OH
Glyceraldehyde3-phosphate
R2
O
H
C
BH
H
Ribose-5-phosphate
CH2O P
C
OH
3
CH2O P
77
CH3
R1
CH2OH
N
C
C
O
HO
C
H
H
C
OH
S
R2
H
B:
3
CH2O P
CH3
R1
N
S
CH2OH
C
O
O
C
H
H
C
OH
Sedoheptulose-7-phosphate
R2
3
CH2O P
C
Carbanion-TPP
78
Coenzyme A
79
Vitamin B5 (Pantothenic Acid)
• Pantothenic acid is also known as vitamin B5
• Pantothenic acid is formed from balanine
and pantoic acid
• Pantothenate is required for synthesis of
CoASH
80
Biosynthesis of CoASH
81
Biosynthesis of CoASH, Cont’d
82
Biosynthesis of CoASH, Cont’d
83
84
85
Function of CoASH
• Since CoA is chemically a thiol, it can react
with carboxylic acids to form thioesters, thus
functioning as an acyl group carrier
• It assists in transferring fatty acids from the
cytoplasm to mitochondria
• A molecule of CoA carrying an acetyl group is
also referred to as acetyl-CoA
• When it is not attached to an acyl group it is
usually referred to as 'CoASH' or 'HSCoA'
86
Acyl Carrier Protein (ACCP)
• 4-Phosphopantetheine moiety, linked via its
phosphate group to the hydroxyl group of
serine, is the active component in another
important molecule in lipid metabolism, acyl
carrier protein
• This is a small protein (8.8 kDa), which is part
of the mechanism of fatty acid synthesis
• However, the final step in fatty acid synthesis
in many types of organism is transfer of the
fatty acyl group from ACP to CoA
87
Acyl Carrier Protein
Thiol group is the point of attachment to the acyl group
being transferred, forming a thioester linkage
88
Structure of CoASH
Thiol group is the point of attachment to the acyl group
being transferred, forming a thioester linkage
Thioester
89
Structure of CoASH, Cont’d
90
Deficiency of Pantothenic Acid
• Deficiency of pantothenic acid is extremely
rare due to its widespread distribution in
whole grain cereals, legumes and meat
• Symptoms of pantothenate deficiency are
difficult to assess since they are subtle and
resemble those of other B vitamin
deficiencies
91
Biochemical Features of CoASH
Acyl transfer reaction
Good leaving
group
Enolization reaction
92
Activation of Carboxylate
Anion by CoASH
93
Activation of Carboxylate Anion,
Cont’d
Good leaving
group
O
R
X
Activation
C
O
Carboxylic acid
R
C
Acy
transfer
Y
R
O Acceptor Y
C
O
Activated
carboxylic group
94
Activation of Carboxylate Anion,
Cont’d
Good leaving
group
OH
R
C
BH
B:
SCoA
R
OH
SCoA
+
H
O
B:
R
C
O
Tetrahedral
intermediate
H2O
C
SCoA
O
Thioester
(Acyl-CoA)
95
Thioesters vs Oxyesters
96
Thioesters vs Oxyesters
• Why thioesters in preference to oxyesters?
• The enzymatic reaction don’t use oxyesters,
but use a thioester derived from CoA
• It is advantageous to use thioesters in
condensation (Claisen) reactions because the
carbonyl carbon atom has more positive
character than the carbonly in the
corresponding oxyesters
97
Thioesters vs Oxyesters, Cont’d
• Thioesters are more readily enolized than
oxyesters
• Thioesters are more “ketonelike” because of
its electronic structures in which the degree of
resonce-eletron delocalization from the sulfur
atom to the acyl group resulting from
overlapping of the occupied p orbitals of
sulfur with the acyl p bond is less than that of
oxyesters
98
Thioesters vs Oxyesters, Cont’d
• The charged-separated resonance form (II) is
a smaller contributor to the electronic
structure in thioesters than in oxyesters
• The reasons for this difference are not fully
understood, but one factor may be the larger
size of sulfur relative to carbon and oxygen,
leading to a poorer energy match for the
overlapping orbitals in thioesters relative to
oxyesters
99
Thioesters vs Oxyesters, Cont’d
• Consider the resonance forms for an oxyester
bellow:
..
..
O
O
O
O
..
..
..
O R
O R
R C ..
O R
R C ..
R C O R
R C +
..
1
I
1
1
1
II
III
• The contribution from form II tends to
decrease the positive charge on the carbon
100
Thioesters vs Oxyesters, Cont’d
• However, for thioester, the contribution form II
is less important, whereas I and III may be
more important than the oxyester
• The carbonly carbon of the thioester is more
positive than that the oxyester
..
..
O
R
C
..
..S
O
R1
R
C
I
..
..S
O
R1
R
O
C S R1
..
II
R
C
+
..
..S
III
101
R1
Thioesters vs Oxyesters, Cont’d
• Positive charge on carbon of the thioester will
make it easier for a nucleophilic compound
such as carbanion to attack the carbonyl
group
• It will also make it easier to remove a proton
from the adjacent carbon atom to form a
carbanion
102
Thioesters vs Oxyesters, Cont’d
Easy to be
deprotonated
H
H
O
C
C
Not easy to be
deprotonated
..
S
..
R
H
Thioester
H
H
O
C
C
..
O
..
R
H
More positive
charge
Oxyester
Less positive
charge
103
Classification of Mechanism of
CoA
104
1. Head Activation Mechanism
(Acyl Group Transfer Mechanism)
• This reaction involving attack of nucleophilic
groups at the acyl carbonyl carbon atom with
transfer of the acyl function to the attacking
group and release of CoA
• This mechanism is called head activation
because the end of acyl function nearest to
the CoA becomes attached to the nucleophile
105
Head Activation Mechanism
(Acyl Group Transfer Mechanism), Cont’d
Good leaving group
O
R
C
O
S
CoA
R
C
S
Nu
+
S
..
Nu
106
CoA
Examples for Head Activation
Mechanism
•
•
•
•
Nu = phosphate: succinly-CoA synthetase
Nu = Amine: glucosamine acyl transferase
Nu = Water: acetyl-CoA hydrolase
Nu = Alcohol: glycerophosphate
acetyltransferase
• Nu = Thiol: lipoate transferase
• Nu = Hydride: acyl-CoA reductase
• Nu = Carbanion: b-ketothiolase
107
2. Tail Activation Mechanism
(Enolization Mechanism)
• This is reaction involving condensation of the
alkyl carbon of the acyl-CoA by the alkyl
carbon by formation of its carbanion
• It is called tail activation because the target
group is attached to the acyl function by the
end furthest from the CoA
108
2. Tail Activation Mechanism
(Enolization Mechanism), Cont’d
• This is reaction involving condensation of the
alkyl carbon of the acyl-CoA by the alkyl
carbon by formation of its carbanion
• It is called tail activation because the target
group is attached to the acyl function by the
end furthest from the CoA
109
2. Tail Activation Mechanism
(Enolization Mechanism), Cont’d
O
O
O
O
C
OH
C
CH3CH
O
C
S
O
..
CH3CH
C
S
Acyl-CoA a-carbanion
CoA
110
CoA
2. Tail Activation Mechanism
(Enolization Mechanism), Cont’d
• The carbanion on the a-C of the propionlyCoA attacks the bicarbonate to make
methylmalonyl-CoA
• The facile character of this reaction is
attributed to the increased acidity of the
thioester compared to the oxyester
• Thioester is 100 – 1000 times more acid
which means that it has a much greater
tendency to undergo proton dissociation at
the methylene function immediately adjacent
to the sulfur
111
2. Tail Activation Mechanism
(Enolization Mechanism), Cont’d
• Negative charge that is produced by this
dissociation is stabilized by delocalization
over the carbonyl group and by the
polarizability of the sulfur
• Example: Citrate synthetase
112
3. Siamese Twin Reaction
(Acyl Transfer and Enolization Mechanism)
• Two molecules of acyl-CoA react together
• One acyl-CoA undergoes head activation and
other undergoes tail activation
• The two important steps of the reaction
depend on both acyl groups being activated,
one for enolization and the other for acylgroup transfer
• In the first step, one of the molecules must be
enolized by the intervention of a base to
remove an a-proton, forming an enolate
113
3. Siamese Twin Reaction
(Acyl Transfer and Enolization Mechanism),
Cont’d
H
H
B:
C
O
C
H
S
H
Acyl-SCoA (Thioester)
CoA
d+
dB H C
O
C
S
CoA
H
Delocalization of the negative
charge
114
3. Siamese Twin Reaction
(Acyl Transfer and Enolization Mechanism),
Cont’d
H
O
C
C
H
S
CoA
H
BH + C
O
C
CoA
S
H
Carbanion enolate
Transition state intermediate
115
3. Siamese Twin Reaction
(Acyl Transfer and Enolization Mechanism),
Cont’d
• The enolate is stabilized by delocalization of
its negative charge between the a-carbon
and the acyl oxygen atom, making it
thermodynamically accessible as an
intermediate
• The developing charge is also stabilized in
the transition state preceding the enolate, so
it is also kinetically accessible that means it is
readily formed
116
3. Siamese Twin Reaction
(Acyl Transfer and Enolization Mechanism),
Cont’d
• If, by contrast, the acetate anion, it would
result in the generation of a second negative
charge in the enolate, an energetically and
kinetically unfavorable process
• Example: b-ketothiolase
117
3. Siamese Twin Reaction
(Acyl Transfer and Enolization Mechanism),
Cont’d
H
H
B:
C
O
C
H
Acetate anion
H
O
d+
B
O
d-
H
C
C
O
H
Unstabilized transition state
118
3. Siamese Twin Reaction
(Acyl Transfer and Enolization Mechanism),
Cont’d
H
O
C
C
H
Acetate enolate
H
O
BH + C
O
C
O
H
Kinetically unfavorable intermediate
119
4. Addition Reaction
• Reactions involving additions to CoA group
• Example: Enoyl-CoA hydratase
120
5. Acyl Group Interchange Reaction
• Reactions involving acyl group interchange
• Example: Acetoacetyl-CoA transferase
121
Mechanism of Succinyl-CoA
Synthetase
(Succinyl Thiokinase )
(Head Activation Mechanism)
122
Reaction of Succinyl-CoA
Synthetase
+
DG˚ = - 2.9 kJ/mol
123
Structure of Succinyl-CoA
Synthetase
• The enzyme is an
a2b2 heterodimer;
the functional units
is one ab pair
124
Mechanism of Succinyl-CoA
Synthetase
(Head Activation Mechanism)
125
Mechanism of Succinyl-CoA Synthetase
(Head Activation Mechanism)
Head activation
SCoA
C
O
O P
OH
Pi
O
His
O
His
(C H2 )2
O
COO
SCoA
OH
H
O
C
O
(CH2 ) 2
COO
N
Succinyl-CoA
O P
N
H
N
N
Tetrahedral
intermediate
It is the displacement of CoA by Pi which generates another high
energy compound, succinly-phosphate (phosphoester)
126
BH
Mechanism of Succinyl-CoA Synthetase
(Head Activation Mechanism)
O
O P
CoASH
O
O
OH
SuccinlylPhosphat
C
O
His
C O P
(CH2 ) 2
COO
His
O
O
N
N
(CH2 ) 2 OH
N
BH
COO
NH
B:
phosphohistidine
His removes the phosphoryl
group with the concomitant
generation of succinate and
phosphohistidine
127
Mechanism of Succinyl-CoA Synthetase
(Head Activation Mechanism)
His
BH
O
O P
O
GDP
GM O P
COO
CH2
OH
O
N
N
phosphohistidine
OH
GDP
CH2
COO
Succinate
128
Mechanism of Succinyl-CoA Synthetase
(Head Activation Mechanism)
His
GTP
H
N
N
129
Mechanism of Citrate
Synthtase
(Tail Activation Mechanism)
130
Citrate Synthase, Cont’d
The monomer of citrate synthase, pictured in the lower frame of the
left side of this screen shows the citrate synthase enzyme bound to
the two products - citrate
131
Reaction of Citrate Synthase
132
• Two binding sites can be found therein:
(1) For citrate or OAA
(2) For CoA
• The active site contains three key residues:
His274, His320, and Asp375 that are highly
selective in their interactions with substrates
• The enzyme changes from opened to closed
with the addition of one of its substrates (such
as OAA)
133
The Active Site of Citrate Synthase
(including His274, His320, and Asp375
134
CS open State
135
CS Closed State
136
Reaction of Citrate Synthase
137
Reaction of CS, Cont’d
OAA
E
E-OAA
Aceyl-CoA
E-OAA-Acyl-CoA
CoA
E-citryl-CoA
Citrate
E-citrate
E
Ordered Mechanism
138
CS Stereochemistry
139
Stereochemistry of the CS
Reaction
140
Stereochemistry of the CS
Reaction, Cont’d
141
Stereochemistry of the CS
Reaction, Cont’d
142
Stereochemistry of the CS
Reaction, Cont’d
143
Mechanism of Citrate Synthase
(Tail Activation Mechanism)
144
Mechanism of CS
Deprotonation of a-H+
Asp 375
C
C
His-320
N
H
COO
C
O
O
O
CH2
H
O
SCoA
N
COO
BH+
C
O
CH2
CoA
O
O
H
N
N
H C H
H C
His 274
H
H
N
N
H C H
N
N
H C O
H
H
SCoA
Enol intermediate
COO
COO
OAA
145
B
Mechanism of CS, Cont’d
• This conversion begins with the negatively
charged oxygen in Asp375 deprotonating
acetyl CoA’s a-carbon
• This pushes the electron to form a doublebond with the carbonyl carbon, which in turn
forces the C=O up to pick up a proton for the
oxygen from one of the nitrogens in of His274
to from enol intermediate
• It is the rate limiting step of the reaction
146
Mechanism of CS, Cont’d
BH+
BH+
C
C
N
O
N
O
H
COO
O
C
CH2
COO
O
H
H
N
N
H
O
H C H
H C O
H
SCoA
Enol
N
H
N
H
COO
B
O
C
CH2
COO
N
H C H
N
H C O
H
SCoA
Carbanion
intermediate
147
BH+
Mechanism of CS, Cont’d
• This neutralizes the R-group (by forming a
lone pair on the nitrogen) and completes the
formation of an enol intermediate
• At this point, His274’s amino lone pair formed
in the last step attacks the proton that was
added to the oxygen in the last step
• The oxygen then reforms the carbonyl bond,
which frees half of the C=C to initiate a
nucleophilic attack to OAA’s carbonyl carbon
148
Mechanism of CS, Cont’d
Hydroxlysis of
citryl-CoA
intermediate
B:
BH+
H
N
C
O H
O
SCoA
O
H C
H
C
SCoA
N
N
H
O
H
C H2
HO
H
N
O
COO
O H
O
N
H
N
H 2O
C
C
O
C H2
HO
C
COO
CH2
CH2
COO
COO
Citryl-CoA (Thioester)
intermediate
Tetrahedral
intermediate149
N
N
Mechanism of CS, Cont’d
• This frees half of the carbonyl bond to
deprotonate one of His320’s amino groups,
which neutralizes one of the nitrogens in its
R-group
• This nucleophilic addition results in the
formation of citroyl-CoA intermediate
• At this point, a water molecule is brought in
and is deprotonated by His320’s amino group
and hydrolysis is initiated
• One of the oxygen’s lone pairs
nucleophilically attacks the carbonyl carbon
of citroyl-CoA
150
Mechanism of CS, Cont’d
• CS entails the formation of a polarized
carbonyl group on OAA and carbanion
formation on Acetyl-CoA enhancing
production of the condensation product, citrylCoA intermediate
• Condensation is followed by the cleavage of
the thioester intermediate within the same
active site to produce citrate
• Each of the important chemical intermediates
in the CS reaction is linked to an enzyme
conformation change
151
Mechanism of CS, Cont’d
BH+
HSCoA
C
N
N
O
O
H
N
H
O
H
C
N
O
C H2
HO
C
COO
CH2
COO
Citrate
152
Mechanism of CS, Cont’d
•
•
•
Why is CS suited hydrolyze citryl-CoA but not acetylCoA?
How is this discrimination accomplished?
CS catalyzes the condensation reaction by bring the
substrates into proximity, orienting them, and
polarizing certain bonds
(1) Acetyl-CoA doesn’t bind to CS until OAA is bound and
ready for condensation
(2) CS conformation changes and creates binding site for
acetyl-CoA
(3) The catalytic residues crucial for the hydrolysis of the
thioester linkage are not appropriately positioned until citrylCoA is formed and this is happened by induced-fit
mechanism to prevent an undesirable side reaction
153