Transcript The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 7 Carboxylations Carboxylations General Concepts • A carbanion (or carbanionic character) must be generated where carboxylation is to occur.

The Organic Chemistry of
Enzyme-Catalyzed Reactions
Chapter 7
Carboxylations
Carboxylations
General Concepts
• A carbanion (or carbanionic character) must be generated
where carboxylation is to occur. Must be a stabilized carbanion.
• Metal ion complexation of the oxygen atom of the keto and
enol forms can increase the acidity of an adjacent C-H bond
by 4-6 orders of magnitude
• CO2 is an excellent electrophile for carboxylation,
but at physiological pH, it is in low concentration
• Predominant form is bicarbonate (HCO3-), which is actually
a nucleophile
• To convert bicarbonate into an electrophile, it must be
activated either by phosphorylation or dehydration
• In general, all enzymes utilize CO2 except for
phosphoenolpyruvate carboxylase and the biotindependent enzymes, which use bicarbonate
• To determine which is the substrate:
Put CO2 into the enzyme reaction at a concentration
approximating its Km value, and incubate with sufficient
enzyme so that a significant amount of product is produced
in the first few seconds. There are two possible outcomes
(Figure 7.1, next slide):
Carboxylations
Test for whether CO2 or HCO3- is the substrate for a carboxylate
CO2 + H2O
H2CO3 (equilibrium ~ 1 min)
nucleophile
electrophile
Possible outcomes when CO2 is added to a carboxylase
B
CO2 as substrate
P R
odr eta
u o
tc f
F
o
r
m
ta
oni
Steady state
Figure 7.1
HCO3- as substrate
P R
odr eta
u
ct fo
F
o
r
m
a
iot
n
A
Steady state
Time
Time
Also, repeat in the presence of carbonic anhydrase
(catalyzes hydrolysis of CO2  H2CO3)
CO2 as Carboxylating Agent
Reaction catalyzed by PEP carboxykinase
Scheme 7.1
OPO3=
H2C
C
COO
-
+ CO2 +
GDP
IDP
ADP
O
Mn2+ or Mg2+
7.1
PEP
-OOCCH
2
C
COO-
+
GTP
ITP
ATP
7.2
oxaloacetate
If run in H218O with CO2, no 18O in products
(need large amount of enzyme so no nonenzymatic conversion of CO2 to HCO3-)
Addition of [14C]pyruvate does not give [14C]oxaloacetate.
Pyruvate or enolpyruvate are not free intermediates.
In the absence of CO2, the enzyme acts like
a kinase (H+ in place of CO2)
PEP carboxykinase-catalyzed reaction
of PEP with ADP (no CO2)
O
PEP + ADP
CH3 C COO7.3
pyruvate
Scheme 7.2
+ ATP
Reduction of Oxaloacetate by Malate
Dehydrogenase
If the carboxylase reaction is run in D2O in the
presence of malate DH/NADH, no D is in the malate;
therefore no enol of oxaloacetate formed.
O
-OOCCH
2
C COO7.2
oxaloacetate
OH
malate dehydrogenase
NADH
-OOCCH
2
CH COO7.4
malate
Scheme 7.3
Malate dehydrogenase traps oxaloacetate
to prevent nonenzymatic enolization.
Hypothetical Mechanism for PEP Carboxykinase
that Involves the Enolate of Oxaloacetate
This mechanism is excluded by the previous result:
OPO3=
B
H
H2C C COO-
=
O PO3
COO-
O
-OOC
PO3= ADP
COO-
ATP
O
-OOC
D+
COO-
-OOC
O
-OOC
COOD
C O
O
-OOC
Scheme 7.4
O
COO7.5
Stereochemistry of the Reaction Catalyzed by
PEP Carboxykinase
Running the reaction in reverse
18O16O
-
S
P
18O-
S
O
O P O P O G
O-
O-
7.6
Scheme 7.5
P
O
O
+
-OOC
COO-
16O-
O
+
CO2 + GDP
COO7.7
inversion of
stereochemistry
Excludes covalent catalytic mechanism
This Mechanism is Excluded:
X
PO3=
O
XPO3=
O
C
O
-OOC
O
GMP O
P OO-
COO-
GTP
COO-
O
Scheme 7.6
Inconsistent with a double-inversion mechanism
for PEP carboxykinase
Possible Mechanism for PEP Carboxykinase
Concerted mechanism for PEP carboxykinase
(or stepwise without release of intermediates)
O
C
O
O
O
CH2 C
C
C
O
O-
O
O
CH2 C
-O
-O
P O-
Mn2+
O
O P
O-
OH2
O P GMP
O O-
Scheme 7.7
O
O
O
C
-O
O-
O
C
O
CH2 C
P
Mn2+
O
O P
O-
OH2
O P GMP
O O-
O-
O
O-
O-
C
O
P
O-
Mn2+
O
O-
O P
O
OH2
O
P
O-
GMP
Reaction Catalyzed by Phosphoenolpyruvate
Carboxytransphosphorylase
Mn2+ or Mg2+
PEP + CO2 + Pi
OAA + PPi
(oxaloacetate)
Scheme 7.8
Same as PEP carboxykinase except Pi
instead of nucleotide diphosphate
All mechanistic experiments are the
same for the two enzymes
Stereochemical Rules Needed to Determine
Stereochemistry of PEP Carboxytransphosphorylase
Alkene stereochemistry nomenclature rules for
(Z)-1-bromo-1-propene (7.8)
c
C
a
b
H
CH3
re
Figure 7.2
H
C
re
C
re Br
7.8
c
b
C
a
re
Alkene Nomenclature Rules for (E)-1bromo-1-propene (7.9)
si
b
CH3
C
a
c
Figure 7.3
C
H si
c
H
C
re Br
7.9
b
C
a
re
si-re or re-si?
Cite the side with the highest priority group
(in this case, Br)
Front face is named re-si face
Two Possible Stereochemical Outcomes for Carboxylation of
PEP Catalyzed by PEP Carboxytransphosphorylase
CO2-O
a
si-re
CO2
-O
CO2-
H
OPO3=
3H
Pi
2C
H
2C
3H
H
(2si,3re)
OPO3
malate
dehydrogenase
O
7.11-R
=
_
H
3 R-[3H]-OAA
CO2-
HO
3H
H
7.10
re-si
3H
H
(Z)-[3-3H]PEP
CO2 (2re,3si)
b
-O
CO2-
3H
H
7.12-(2S,3R)
O
2C
(2S,3R) malate
CO2
-
7.11-S
H
_
fumarase
3-S-[3H] OAA
malate
dehydrogenase
(E)-[3-3H]PEP,
3H
With
98%
in fumarate; therefore
carboxylation from si-re face
-O
2C
H2O +
H
3H
antieliminationH
CO2-
P-O bond of PEP breaks, but
7.13-3H
C-O bond of PEP breaks with
EPSP synthase
Scheme 7.10
fumarase
-H2O
3H
CO2-
2C
OH
7.12-(2S,3S)
(2S,3S) malate
H
+
H
CO2-
H
-O
antielimination
3HOH
CO27.13-1H
fumarate
observed
98% loss
as 3H2O
Vitamin K Cycle for Carboxylation of Proteins
O
R=
R
blood-clotting
proteins
O
7.14
vitamin K
reductase
O
NH
O
NH
NH
COOH
Glu
Gla
COOH
OH
NH
COOH
vitamin K carboxylase
CO2, O2
R
OH
7.15
O
vitamin K epoxide reductase
O
R
RSH
Scheme 7.11
O
7.16
binds
Ca2+
Calcium-dependent Binding of Clotting
Proteins to Cell Surfaces
Figure 7.4
=
O3PO
NH
O
COO
HN
Ca2+
Gla
O
COO
=
O3PO
membrane bilayer
=
O3PO
R
NH
clotting proteins
=
O3PO
-proteases
cell surface
Holds the proteases to the appropriate cells,
triggering the blood-clotting cascade
Test for Carbanion vs. Radical
Mechanisms for Vitamin K Carboxylase
Scheme 7.12
NH
CO
_
CO
erythroand threo-
COO-
COO-
F
COO-
CO
carbanion
F
NH
NH
radical
NH
F
CO
NH
•
F
COO-
CO
COOCOO-
erythro- F- elimination, but not threo-;
therefore stereospecific (carbanion)
Stereochemical Outcome of Vitamin K
Carboxylase-catalyzed Carboxylation of
(2S,4R-fluoroglutamate)
O
O
Phe Leu NH
Glu Val
H
vitamin K
carboxylase
Phe Leu NH
H
F
H
-OOC
Glu Val
F
7.17
Scheme 7.13
-OOC
COO-
carboxylation with
inversion of
stereochemistry
Proposed Vitamin K Carboxylase-catalyzed
Carboxylation of Glutamate Residues via a
Carbanionic Intermediate
O
*
H
O C O-
H
*
H
§C
:B
+
§
*
H
-OOC
O
O
O
+
C
O
+
O
Scheme 7.14
But where does vitamin K fit into the mechanism?
Model Study for Function of Vitamin K
Chemical model study for the activation of
vitamin K1 as a base
Not a strong enough base to deprotonate 7.20
Model for reduced
vitamin K
O - K+
O
O
_
O2
O
O O-
O
7.18
O
C
Reaction does not work in
absence of O2
OEt
O
O
CO2Et
7.21
H
EtO2C
7.20
Dieckmann
condensation
O
_O
7.19
strong
base
Scheme 7.15
Base Strength Amplification Mechanism
Two Proposed Mechanisms for Activation
of Vitamin K1 as a Base
-B
OH
R
O
O
HO
O
O
HO
O-
O-
HO
O
-HO-
O
CH3
CH3
O
7.22
(not 1O2)
R = phytyl
R
O
CH3
CH3
CH3
O
R
R
_
R
O2
A
OH
O
O
O
may pull off
-proton
from Glu
residues
-B
H
O
O
R
B
CH3
OH
R = phytyl
O
O
O2
O
R
O
O
-H+
+H+
O
R
OH
O
CH3
O
CH3
OH
O
B-
H
R
O
+
HO-
CH3
Scheme 7.16
When run in 18O2, 0.95 mol atom 18O in epoxide
0.17 mol atom 18O in quinone oxygen
To Determine Which Ketone is Involved
18O
O
phytyl
phytyl
CH3
CH3
18O
O
7.23
7.24
Incubation in 16O2 atmosphere gives loss of
0.17 mol atom 18O from 7.23, none from 7.24
Therefore, the ketone next to the methyl group
is involved in the reaction
Modified Base Strength Amplification Mechanism
for Vitamin K Carboxylase
To account for much loss of 18O from substrate
O
-S
H
weak base
O-
O
phytyl
phytyl
H18O
18OH
O-O-
phytyl
O
H18O
O
O2
O
phytyl
O
O
O
18O-
OH18O
7.26a
O
Scheme 7.18
phytyl
OH
7.26b
O
phytyl
phytyl
O
O
O
+ H18O-
18O
strong base
+ HO-
Bicarbonate as the Carboxylating Agent
Reaction catalyzed by PEP carboxylase
O
O
CH2
P
OO-
C
COO-
Mg++
+ HC18O3-
PEP
Scheme 7.19
2 (18O)
-OOC
1 18O atom
O
CH2C
COO-
+
Pi(18O)
7.27
No H218O formed
(high enzyme concentration,
short time at alkaline pH)
Therefore HCO3-, not CO2
Concerted (A), Stepwise Associative (B), and Stepwise
Dissociative (C) Mechanisms for PEP Carboxylase
Note: nucleophilic mechanisms
O-
18
-O
A
18
18
C
O
P
-O
O
Scheme 7.20
O18
-O
18
O
18O
O-
C
18
HO
O
P
O
O
18 O
O-
B
O
P
O
HO
O-
C
O
O
O
H
B:
COO-
COO-
-O
18 O
P
18
stepwise
associative
O
18
-O
COO
O-
18
HO
Pi (18O)
Mg2+
-O
18
18O
O
O-
COO18 O
O-
P
18
18
O
COO-
-O
18O
O-
18
HO
COO-
-O
18
concerted
O
18
-O
HO
COO-
Pi (18O)
18
O-
P
O
O
O
Mg2+
COO
18O
C
18 O
18O
Pi (18O)
18
-O
O
Mg2+
COO
O
stepwise
dissociative
COO-
No partial exchange detected ([14C]pyruvate does not give [14C]PEP)
Therefore, either concerted or intermediate not released
Evidence for Stepwise Mechanism
17O
16O
S
P
S
O
in H2
COOH
7.28
18O
18O
P
17O
inversion
16O
7.29
concerted is suprafacial
sigmatropic; therefore retention
Also, rate is independent of pH, but the carbon isotope
effect for H13CO3- decreases with increasing pH. Not
possible with concerted
Evidence for dissociative mechanism:
Using methyl PEP and HC18O3- more than 1 18O in Pi
and substrate recovered has 18O in nonbridging position
of phosphate; therefore reversible CO2 + Pi formed (see
next slide)
Mechanism for Incorporation of 18O into Substrate
18 O
-O
18
O-
C
O
O
P
H
O
-O
-O
18 O
O-
18
HO
-O
18
O-
P
18
O
18O
Mg2+
B:
COO-
O
C
O
O
O-
P
18
COO
O
18 O
B
Mg2+
H
COO
Non-bridging 18O
-O 18
18 O
O
H
O-
P
18
O
O
18 O
COO
O-
O-
18
HO
O
O
P
O
Mg2+
B:
-O 18
18
18 O
O-
18
more than one 18O
incorporated into PEP
COO-
HO
Scheme not in text (after Scheme 7.20)
Note: the ultimate carboxylating agent is CO2
Biotin-dependent Enzymes
Multisubunit enzymes
Covalent attachment of d-biotin to an active site
lysine residue
O
O
HN
ATP
NH
H
AMP + PPi
HN
NH
H
H
H
(CH2)4COOS
S
H
N
H
H
7.35
7.34
142 o 118 o
S
-OOC
H
H
H
N
NH
O
HN
O
O
Scheme 7.24
H
7.34
Enzyme reactions with HC18O3- give Pi with one 18O
and product with 2 18O atoms (bicarbonate)
Reactions Catalyzed by Biotin-dependent
Carboxylases
Figure 7.5
O
O
ATP + HCO3- +
H3C C
COO-
-OOCCH
2
C COO-
+ ADP + Pi
Pyruvate carboxylase
O
ATP + HCO3- +
O
-OOCCH
2
H3C C SCoA
+ ADP + Pi
C SCoA
Acetyl CoA carboxylase
COO- O
O
ATP + HCO3- +
CH3CH2 C SCoA
C SCoA + ADP + Pi
CH3 CH
Propionyl CoA carboxylase
COO-
O
ATP + HCO3- +
O
SCoA
SCoA
+ ADP + Pi
b-Methylcrotonyl CoA carboxylase
NH2
N
N
N
N
O
O
H3C
CH3 O
O P O P O
-O
O-
OH OPO3=
O
N
H
N
H
SH
HO H
CoASH
Diagnostic method for biotin - add avidin
KD = 1.3  10-15 M
Mechanism of Biotin-Dependent Carboxylases
Partial exchange reaction of 32Pi into ATP (in absence of
substrate) with biotin-dependent carboxylases
ATP +
32P
M+2
HCO3ADP
i
AT32P + Pi
Scheme 7.25
No substrate or product needed
Suggests ATP activates bicarbonate
Mechanism for Partial Exchange of 32Pi into
ATP with Biotin-dependent Carboxylases
O
O
O-
HO
+ ATP
O
HO
+ ADP
HO
O
-HPO43OPO3=
H32PO4=
Scheme 7.26
OPO3=
O
ADP
HO
O-32PO3=
HO
O-
+ AT32P
Partial Exchange Reaction of [14C]ADP into
ATP with Biotin-dependent Carboxylases
[14C]-ADP + ATP
Scheme 7.27
HCO3-/M2+
[14C]-ATP + ADP
Mechanism for Partial Exchange Reaction
of [14C]ADP into ATP with Biotindependent Carboxylases
ATP
HCO3-
O
HO C O PO3=
[14C]ATP
+ ADP
[14C]ADP
Scheme 7.28
[14C]product
substrate, HCO3ATP, M2+
[14C]substrate
(reaction is reversible)
Evidence for Enzyme-Bound Intermediate
In the absence of pyruvate get a carboxylated enzyme
Pyruvate carboxylase-catalyzed incorporation of 14C
from H14CO3- into the enzyme
Scheme 7.29
O
O
HO
14 O-
if pyruvate HO C
14
is added
+ ATP
M2+
HO C
-X
O
+
:B O
H2
H C C COOH
14
X
+ Pi
+
ADP
O
HOO14C CH2 C COOH
+
-X
X
Carboxylated enzyme is unstable to acid (pH 4.5), but
stable to base (0.033 N KOH)
[14C] carboxylated enzyme in base purified by gel filtration
then stabilized by CH2N2 treatment (makes methyl ester)
Isolation of N1-methoxycarbonylbiotin from the
Reaction Catalyzed by Pyruvate Carboxylase Followed
by Diazomethane Trapping of the N-carboxybiotin
O
CH3OC N
O
NH
O
C
O
NH(CH2)4 CH
S
N
H
O
CH3OC N
Lys
trypsin
papain
HN
The X in previous Scheme
Scheme 7.30
C
O
O
biotinidase
NH
+
O
CH3OC N
O
NH
COOS
7.36
Isolated; X-ray
crystal structure
S
O
NH(CH2)4 CHCOONH3+
Six Possible Mechanisms for Formation of N1-carboxybiotin
O
1.
HOC OPO3=
HCO3- + ATP•Mg2+
+ ADP•Mg2+
Figure 7.6
O
O C OPO3=
CO2
O
O
O
PO43-
C
H N
NH
PO43-
N
O
O
O
NH
HO
C N
NH
H
R
S
S
B
R
S
R
7.37
O
O
=
Mg2+ O3P O P O P OAdo
2.
O-
O
O
O-
=O P
3
B:
H N
NH
N
NH
-ADP
R
S
O
O
-O C
S
O
R
N
O
B:
3.
H N
NH
S
R
O
NH
NH
-PO44-
OH
R
S
R
7.37
O
Mg2+ =O3P O P O P OAdo
OOO
HCO3- =
O3P
O
N
NH
-ADP
N
NH
S
N
OH
S
O
PO3=
O
O
HO
stepwise
ON
NH
7.37
S
R
S
R
OPO3=
R
4.
O
O
B:
H N
NH
N
HO
C
NH
O-
O
R
S
R O-
S
O-
PO3= 2+
Mg
O
AdoO P O P
O
O
H
B
B:
H N
HO
Mg2+ =O3P
C
O
R
S
N
O
NH
O-
O-
O C
=
Mg2+ O3P
OH
S
PO3= Mg2+
O
Pi
NH
7.37
O
S
O
ADP
NH
N
R
S
O
O-
NH
O
O
5.
OH
HO C N
O-
-H+
ADP
O
R
Pi
N
NH
7.37
OH
R
R
S
AdoO P O P O
O
O
OMg2+
=O P
3
O P O P OAdo
O
O
6.
O-
B:
O
ADP
H N
NH
O
-O
O
C
O
H
B
O-
O
N
Figure 7.6
R
OH
O
NH
OH
O
-O
P
pseudorotation
O
N
O-
HO
P
O
OH
S
-O
P
Pi
O
O
NH
O-
7.37
:N
NH
OH
S
R
S
R
S
R
In the presence of HCO3- but absence of biotin, biotin carboxylase catalyzes
hydrolysis of ATP; with HC18O3- one 18O incorporated into Pi; therefore
supports formation of carboxyphosphate (mechanism 1).
Mechanism for the Formation of
Carboxyphosphate in the Reaction
Catalyzed by Acetyl-CoA Carboxylase
carboxyphosphate
HC18O3-
O
-O
O
O
P O P O P OAdo
OOO-
18O
H18O
18O
PO3=
H2O
Scheme 7.31
[18O] Pi + C18O2
+ ADP
A
Possible Mechanisms for Transfer of CO2
from N1-carboxybiotin to Substrates
Concerted
O
H
-OOC
CH2
O
O
-OOC
N
-OOC
NH
CH2
COO-
S
B
R
Stepwise-associative
O
O
-OOC
-
CH2
B
-OOC
O
CH
_ 2
-OOC
N
CH2
COO-
R
Stepwise-dissociative
O
-OOC
C
O
CH2
-OOC
O
-OOC
CH
_ 2
O H
O
O
-OOC
NH
S
C
associative
O
H
N
COO-
NH
S
CH2
R
dissociative
O
C
Figure 7.8
O
Initial evidence for concerted:
retention of configuration at -carbon
Evidence for Stepwise Mechanism
Transcarboxylase and propionyl-CoA carboxylasecatalyzed elimination of HF from -fluoropropionyl-CoA
O
O
CoAS
B-
F
+ HF
CoAS
H
7.44
7.43
Scheme 7.37
Double isotope fractionation test:
O
O
Compare 13CH3COOH with 13CD3COOH
If concerted, should show both 2H and 13C isotope effects (C-H bond
broken and C-C bond made simultaneously)
If stepwise, not necessarily so
Also, if stepwise, 13C isotope effect could be different with
and without 2H
13(V/K) for 13CH COCOOH
1.0227
3
13(V/K) for 13CD COCOOH
1.0141 (calculated value is 1.0136)
3
therefore stepwise