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The Organic Chemistry of
Enzyme-Catalyzed Reactions
Chapter 8
Decarboxylation
Decarboxylation Reactions
Driving force for decarboxylation
O
R
CH2 C
stabilized
O
R CH2
+ CO2
RCH3
O
R
CH
CH2
R
C
O
X
Scheme 8.1
CH
CH2
+ X- + CO2
Decarboxylation of -Keto Acids
O
R
C
O
CH2
8.1
O
O
-CO2
C
R
C
CH2
R
C
O
CH2
+H+
O-
Scheme 8.2
Decarboxylation is accelerated in acid
R
CCH3
Cyclic Transition State for
Decarboxylation of -Keto Acids
H
O
O
R
OH
O
R
CH2
+ CO2
8.2
Scheme 8.3
Strong acids are needed to protonate carbonyls (pKa -7)
Protonation of Imines (pKa about +7) is Easy
Amine-catalyzed decarboxylation of -keto acids
O
R
C
CH2 COO-
+
R' NH2
R
O
C
CH2
C
H2O
O-
NH
R'
+
8.3
CO2
R' NH2
+
H2O
R
C
O
Scheme 8.4
CH3
R
C
CH3
R
C
NH
: NH
R'
R'
CH2
H
OH2
Reaction Catalyzed by Acetoacetate
Decarboxylase
CH3 C CH2COOO
8.4
Scheme 8.5
CH3 C
O
CH3
+ CO2
Schiff Base Mechanism
Fate of the ketone oxygen in the reaction
catalyzed by acetoacetate decarboxylase
Suggests a
Schiff base
mechanism
Scheme 8.6
18O
H3C C CH2COO-
acetoacetate
decarboxylase
H2O
O
H3C
C
CH3
+ CO2
+
In D2O, D is incorporated into acetone
pKa of Lys-115 is 5.9; adjacent to Lys-116,
which lowers pKa by about 4.5 pKa units
Why?
H218O
Reaction of the K115C Mutant of Acetoacetate
Decarboxylase with 2-Bromoethylamine
K115
K115C
Br
NH3+Br
S
SH
inactive
mutant
NH3+
Scheme 8.7
NH3+
8.5
after aminoethylation
it is active
Lys-116 mutants are still active (but less than WT)
pKa of Lys-115 in Lys-116 mutants is >9
Aminoethylation of K116C - lowers pKa of K115
back to 5.9
Schiff Base Mechanism
Proposed mechanism for acetoacetate decarboxylase
D
D
B
18O
CH3C
18OD
CH2COO-
CH2COO-
CH3C
D
N
:
Lys115
B
ND2
Lys115
-D218O
O
CH3C
CH2
ND
:B
D
C
O-
8.6
Lys115
- CO2
D
CH3
C
CH2D
D2 O
CH3
C
CH2D
CH3
C
:
Lys115
ND2
O
+
ND
Lys115
8.8
Scheme 8.8
: ND
Lys115
8.7
CH2
B
Test for Schiff Base Mechanism
NaBH4 reduction during the reaction catalyzed
by acetoacetate decarboxylase
H
H
CH3
14C
CH3
NH
CH3
14C
CH3
NaBH4
+
+
Lys115
Lys115
8.9
isolated
O
NH
CH3
NH2
-CO2
CH314C
14C

NH
Lys115
8.8
H3
CH3
O+
CH2
NaBH4
C
8.6
Lys115
H
O-
CH314C CH2
O
C
O-
H3O+

H
CH314C CH2
NH2
Lys115
8.10
Scheme 8.9
C
O-
NH2
Lys115
O
Metal Ion-catalyzed Mechanism
Alternative to Schiff base mechanism
D
R
O
18O
M++
O-
Scheme 8.10
R
O
18O
O
M++
- CO2
B
R
R
CH2D
18O
18O
M++
M++
no loss of
carbonyl
oxygen
With 14C substrate + NaBH4 no 14C protein
Proposed Mechanism for the Decarboxylation
of (S)--acetolacetate (8.11) Catalyzed by
-Acetolactate Decarboxylase
Me
H
OH
Me
O
O
Me
O
OH
Me
O
Mg2+
8.12
8.11
-CO2
B
H
Me
OH
O
Me
Mg2+
Scheme 8.11
inversion of
stereochemistry
-Hydroxy Acids
Proposed mechanism for the isocitrate
dehydrogenase-catalyzed conversion of
isocitrate (8.17) to -ketoglutarate (8.19)
CH2COO-
CH2COOH
C
COO+
H
C
NADP+
COO-
M++
H
C
C
NADPH +
-OOC
CH2COO-
H
CH2COO-
H
O
C
-CO2
CH2
O
C
OH
O
8.17
8.18
isocitrate
B
M++
oxalosuccinate
-OOC
O
M++
C
O
-OOC
M++
8.19
-ketoglutarate
Scheme 8.15
Oxalosuccinate is not detected.
Also no partial reactions, but it is presumably formed
Not All Decarboxylations Need Schiff
Base or M2+
Reaction catalyzed by phosphogluconate
dehydrogenase
COOScheme 8.16
H
C
OH
CH2OH
HO
C
H
C
O
H
C
OH
H
C
OH
H
C
OH
H
C
OH
+ NADP+
CH2OPO3=
CH2OPO3=
8.20
8.21
6-phosphogluconate
+ CO2 +
ribulose 5-phosphate
Experiments?
NADPH
Proposed Mechanism for the Reaction
Catalyzed by Phosphogluconate
Dehydrogenase
H
O-
O
C
H
H18O
C
C
O-
O
NADP+
OH
H
B
R
8.20
Scheme 8.17
H
C
NADPH
H
H
C
18O
C
R
8.22
B
OH
C
OH
CH2 OH
-CO2
18O
C
18O
B:
H
R
C
R
8.21
-Keto Acids
Improbable decarboxylation of -keto acids
O
OR
O
-CO2
R
O
8.23
Scheme 8.18
8.24
Cofactor Required
Diphosphorylation of thiamin
Scheme 8.19
CH3
NH2
N
H3C
ATP
OH
N
AMP
4'
3' N
H
1
8.25
H3C 2' N
1'
6'
O
5
5'
S
N
CH3
NH2
O
N
H 2
4
S
3
8.26
thiamin
(vitamin B1)
thiamin diphosphate
vitamin
coenzyme
P
O-
O
O
P
O-
O-
Abbreviated Form for TDP
R
R'
N
H
2
S
8.27
Resonance Stabilization of Thiazolium Ylide
R
R'
N
R
R'
N
S
S
R
R'
N
S
R
R'
N
S
H
B
8.28
exchangeable in
neutral D2O at room
temperature
pKa estimated 13-18
8.29
8.30
Scheme 8.20
Proposed Mechanism for Autodeprotonation of
C-2 of Thiamin Diphosphate
418Glu
O
418Glu
O
H 1'
N
O
N
N
3'
4'
NH
O
OP2O63-
H
N
N
S
OP2O63-
N
NH
S
H
H
:B
Without N-1 N or N-4 NH2
it is not active
Without N-3 N it is active
418Glu
O
OH
N
OP2O63-
N
N
NH2
S
Scheme 8.21
Stable, but C-2
proton not very
acidic
Me
+
N
Me
+
N
S
H
8.41
Ideal
heterocycle
Me
+
N
O
H
8.42
NH
H
100 times more acidic, but
does not catalyze -keto
acid decarboxylation and is
easily hydrolyzed at pH 7
8.43
Mechanism of Thiamin Diphosphate-dependent Enzymes
Nonoxidative decarboxylation of -keto acids: (A) the reaction
catalyzed by pyruvate decarboxylase, (B) the reaction catalyzed by
acetolactate synthase
O
A
H3C C COOH
CH3CHO + CO2
pyruvate decarboxylase
8.44
O
B
2 H3C C COOH
O
H3 C C
CH3
C
COO-
+ CO2
acetolactate synthase
OH
Scheme 8.26
8.45
acetolactate
decarboxylase
O
H3C C
CO2
CH3
C
OH
8.46
acetoin
H
Benzoin Condensation
Chemical model for formation of 8.45
O
2
C H
O
CN
OH
8.47
Scheme 8.27
Mechanism for the Benzoin Condensation
nucleophile
H
:B
B
H
O
C H
HO
C
C
N
H
O
H
B
B:
HO
C
C
N
H
O
H
CN
CN OH
8.48
8.50
8.49
electrophile
Scheme 8.29
HO
C
C
O
N
CN
+
catalyst
OH
8.47
Proposed Mechanism for the Reaction
Catalyzed by Acetolactate Synthase
like -CN
R'
R
R'
N
R
R'
N
R
S
R'
-CO2
N
S
R
R'
N:
R
S
N
S
S
O
H
CH3
B:
CH3
C
C
C
COOOH
H
OH
OH
CH3
8.51
B+
C
CH3
8.52
O
electrophile
CH3
O-
nucleophile
C
COO-
O
B+
H
R'
O
C
CH3
C
CH3
OH
8.46
acetolactate
decarboxylase
H
-CO2
CH3
O
CH3
C
C
R
S
COO-
:B
OH
8.45
R'
R
N
S
Scheme 8.30
N
catalyst
CH3
C
O
CH3
C
C
OH
8.53
H
O
O-
Examples of Oxidative Decarboxylation
of -Keto Acids
O
A
H3C
O
COO-
H3C
+ CoASH
SCoA
+ CO2
O
B
-OOC
O
COO-
-KG
+ CoASH
-OOC
SCoA
succinyl-CoA
Scheme 8.33
Multienzyme complexes
5 different coenzymes involved
+ CO2
-Keto Acid Dehydrogenase
Proposed mechanism for the reaction catalyzed
by dihydrolipoyl transacetylase
dihydrolipoyl
transacetylase
pyruvate
decarboxylase
H
R'
+
R N
CH3
TDP
S
C
_
R'
B
NH
R
C
S
+
Lys
+
N
S
:B
O
S
CH3
8.57
C
OH
O
H
S
lipoic acid
SH
8.52
-CO2
8.58
O
O
CH3C
SCoA
O
8.61
CH3CCOOH
SH
:
+
FAD
R
CoASH
SH
CH3
R'
C
S
SH
+
R
+
N
S
_
lipoic acid
NAD+
R
R
8.59
8.62
reduced lipoic acid
acetyl lipoamide
dihydrolipoyl dehydrogenase
Scheme 8.34
CH3
CH2 C
CH C NH CH2 CH2 C NHCH2CH2SH
CH3 OH
O
O P
O
O
NH2
ON
O
O P O CH2 N
O
O=O PO
3
N
N
OH
8.60
Coenzyme A
Alternative Proposed Mechanism for the
Reaction Catalyzed by Dihydrolipoyl
Transacetylase
R'
R'
R
+
N
R'
+
R N
S
+
R N
S
:B
CH3
C
S
O
H
H
B+
HSCoA
O
+
+
SH
O
SH
CH3C
R
R
Scheme 8.35
_
-H+
CH3
SH
S
SCoA
Proposed Mechanism for the Reaction
Catalyzed by Dihydrolipoyl Dehydrogenase
dihydrolipoyl
dehydrogenase
R
N
B:
O
R
N
N
NH
N
H
SH
N
O
S
O
S
R
N
S
+
NH
N
H
R
O
S
S
_
N
N
H
NH
O
H
NAD+
:B
R
O
R
NADH
8.62
Scheme 8.37
Flox
Amino Acid Decarboxylation
covalently bound
via Schiff base to
a Lys residue
Conversion of pyridoxine to
pyridoxal 5-phosphate (PLP)
CH2OH
CHO
OH
HO
=O
N
CH3
8.63
pyridoxine
(vitamin B6)
OH
3PO
N
8.64
Pyridoxal 5-phosphate
(PLP)
coenzyme
Scheme 8.38
CH3
The First Step Catalyzed by All PLP-dependent
Enzymes, the Formation of the Schiff Base between
the Amino Acid Substrate and PLP
Lys
H
HN+
=O
..
NH2
R
OH
=O
3PO
:B
Lys
B+
H
.. H
+N
HN
H
COO-
OH
3PO
+N
H
COOR
+N
H
8.65
Lys
NH2
+BH
:B
H
COO-
Lys
R
H2N+
NH
=O
OH
3PO
+N
H
8.66
=O
+BH
:B
..
N
H
COO-
OH
3PO
R
+N
H
Scheme 8.39
Schiff Base Formation Increases the
Electrophilicity of the Carbonyl
(A) Reaction of an amine with an imine
(B) Reaction of an amine with an aldehyde
A
R
NHR1
B
R
O
+
+
Scheme 8.40
R2NH2
R
NHR2
+
R1NH2
R1NH3+
R
NHR1
+
H2O
30x faster
than
Reaction Catalyzed by PLP Decarboxylases
(different enzymes for different amino acids)
NH3
R
COO
NH2
PLP
decarboxylase
R
neutralizes
acidic conditions
Scheme 8.41
+
CO2
increases intracellular
pressure
To Provide Evidence for a Schiff Base
with an Active Site Lysine Residue
Reduction and hydrolysis of PLP enzymes.
Lys
Lys
+
NH
+
NH2
OH
=O PO
3
1. NaBH4
OH
=O PO
3
2. hydrolysis
N+
H
N+
H
8.67
Scheme 8.42
If Substrate Is Added before NaBH4
R
COOH
COOH
R
+
NH +
NH2
NaBH4
=O
OH
3PO
N+
H
OH
=O PO
3
N+
H
8.68
No 18O from H218O Found in CO2
Incorrect hydrolytic mechanism for
PLP-dependent enzymes
H
R
O
C
+
H H
C
R
O-
+ HCO3-
C
+
NH
B
RCH2
NH
OH
=O
OH
3PO
N+
H
Scheme 8.43
CO2
+
NH
=O
OH
3PO
N
H
=O
OH
3PO
N+
H
+
OH
Proposed Mechanism for PLP-dependent
Decarboxylases
stereospecific
incorporation
of proton
Lys
Lys
+
=O
NH
H
H
O
R C C O-
+
NH
R C COONH3+
N
=O
+H
OH
3PO
H
NH
+ NH
-CO2
=O
OH
3PO
N
H
electron sink to
stabilize anion
R
CH2
Lys
=O
OH
3PO
N
+H
Scheme 8.44
+
Lys
NH
NH2
+ RCH2NH2
=O
=O
OH
3PO
N
H
:
N
H+
+ NH
R CH
R C
+
H
OH
3PO
+B
NH2
OH
3PO
N+
H
O
Pyruvoyl-Dependent Decarboxylases Identification and Differentiation
OH
OH
O
1.5 N
HCl 100 °C
OH
1. NaB3H4
O
NH
a
NH3
R
NH
H3O+ / 
OH B+
NH
O
R
NH
-CO2
NH
-O
NH
NH
R
O
NaBH4
b
H
N
NH2
O
O
2. 6 N HCl
110 °C
NH3
NaCNBH3
Proposed mechanism for pyruvoyl-dependent
decarboxylases (amino acids)
O
COO+
O
H3
NaBH4
NH
R
O
NH
O
H3O+ / 
Scheme 8.45
NH
c
COO-
R
R
NH
H3O+ / 
COOH
NH3
COOH
alanine
NH2
COOH
8.69
NH2
R
R
COOH
8.70
PLP and PQQ enzymes have absorbance >300 nm
Pyruvoyl enzymes - no absorbance >300 nm
PLP and PQQ enzymes do not give the products shown above
Differences
Inactivation of S-Adenosylmethionine
Decarboxylase by its Substrate
+
NH
H
normal
turnover
O
O
COO+
NH
NH
B H
+
NH3
O
O-
O
-CO2
S Me
NH
S Me
-O
S-adenosylmethionine
(SAM)
S
S
O
NH
Scheme 8.46
S Me
NH
Ado
NH
Me - MeSAdo
Ado
O
NH
B:
S
C140
8.72
8.73
NH
H2O
O
NH
S
C140
8.75
NH
O
NaBH4
C140
8.76
S
H
NH2
trypsin digestion
O
+
NH
inactivation
OH
+
NH3
+
Me
O
8.71
O
Me
Ado
NH
Ado
NH
Ado
Ado
O
+
NH
S
S
C140
8.74
Biosynthesis of the Active-site Pyruvoyl
Group of Histidine Decarboxylase
prohistidine decarboxylase
18OH
B:
O
N
H
18OH
b
NH
H
O
C
C
a
B:
O
N
H
NH
18O
b
18OH
NH
:
NH2
a
Ser 81
O
H
Ser 82
b
18OH
18OH
O
N
H
NH
O
NH
H18O-
O
Ser 81
both
Scheme 8.47
18O-
NH
18O
O
O
NH
NH2
H2 O
CH3
NH
O
Ser 82
end up here
Pathway b is valid only if the hydroxide released is the same one
that hydrolyzes the amide bond.
Other Decarboxylations
Proposed addition/elimination for orotidine 5monophosphate decarboxylase
O
O
-X
X
HN
O
O
H
HN
N
C
R
O
OH
Scheme 8.48
O
OH
N
R
B+
O-
X
HN
O
O
O
N
R
- CO2
O
H
:B
HN
O
N
R
Proposed Zwitterion Mechanism for Orotidine
5-Monophosphate Decarboxylase
O
O
HN
HN
- CO2
O-
:
O
B+
H
N
COO-
R
HO
N
R
O
O
+
O
HN
HN
HO
N
R
H O
+
H
B:
B
Scheme 8.49
O
HN
N
R
+
O
N
R
Model Study for the First Mechanism
Model reactions for the addition/elimination mechanism for
orotidine 5-monophosphate decarboxylase
O
O
NaHSO3
MeN
SO3Na
MeN
A
O
SO3Na
R
N
Me
O
N
Me
OH
O
O
O
SMe
MeN
B
O
Me
MeI
H
AgBF4
OH
N
Me
R
O
Scheme 8.50
MeN
O
O
+
SMe2
Me
-CO2
H
-Me2S
OH
N
Me
O
Me
MeN
O
N
Me
H
Support for the Second Mechanism
(actually, disproof of the first mechanism)
O
O
13
HN
O
N
D
HN
O-
O
N
R
R
8.77
8.78
incubate with enzyme - no
rehybridization by 13C NMR
COO-
no secondary
deuterium
isotope effect
More Evidence Against the First Mechanism
O
3PO
O
HN
N
COO–
O
OH
O
X
HN
=O
O
=O PO
3
O
N
N
O
OH
8.79
X = Br, Cl inhibitors
X=F
substrate
OH
HN
COO–
=O
3PO
O
N
N
O
OH
8.80
excellent
substrate
OH
OH
8.81
no rehybridization
O
HN
O
N
R
Me
8.82
When R = COOH
OMP decarboxylase is the most proficient enzyme known
kcat = 39 s-1
knon = 2.8  10-16 s-1
(nonenzymatic)
kcat/knon = 1.4  1017
(rate enhancement)
Rationale for Direct Decarboxylation
O
O
HN
HO
O
HN
HO
N
H
HN
N
H
HO
N
H
original
proposal
8.83
protonation
at O-2
OH
protonation
at O-4
OH
HN
HN
O
O
N
H
N
H
based on
calculations
8.84
Two atoms of Zn2+ in active site may stabilize negative charge
Reaction Catalyzed by Mevalonate
Diphosphate Decarboxylase
Decarboxylative elimination
H3C
OH
CH3
O
O
O-
O
O P O P OO-
+ ATP
O
O-
8.85
Scheme 8.51
O P
8.86
O-
O
O P OO-
+ CO2 +
ADP +
Pi
F
OH
OH
O
O
O8.87
O P O P OO-
O
O
O-
O
O8.88
O
O P O P OO-
Both are poor substrates
Destabilize a carbocation intermediate
O-
Very Potent Inhibitor - TS‡ Analogue Inhibitor
H3C
+ H
N
mimics a carbocation
O
O
O-
O P
O-
8.89
O
O P OO-
Proposed Carbocation Mechanism for
Mevalonate Diphosphate Decarboxylase
OH
ATP
O
O-
OPP
Scheme 8.52
OPO3H-
ADP
O
+
-HPO42O-
OPP
O
O-
-CO2
OPP
8.86