the organic chemistry of enzyme-catalyzed reactions chapter 7 carboxylations
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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.
• 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
Must be a stabilized carbanion.
• In general, all enzymes utilize CO2 except for
phosphoenolpyruvate carboxylase and the biotin-dependent enzymes, which use bicarbonate
• To determine which is the substrate: Put CO2 into the enzyme reaction at a concentrationapproximating 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):
Figure 7.1
Carboxylations
electrophileCO2 + H2O H2CO3
nucleophile
(equilibrium ~ 1 min)
Possible outcomes when CO2 is added to a carboxylase
Also, repeat in the presence of carbonic anhydrase (catalyzes hydrolysis of CO2 H2CO3)
Test for whether CO2 or HCO3- is the substrate for a carboxylate
Steady state
TimeTime
Steady state
A B
CO2 as substrate HCO3- as substrate
Scheme 7.1
CO2 as Carboxylating Agent
PEPoxaloacetate
If run in H218O with CO2, no 18O in products
Reaction catalyzed by PEP carboxykinase
+ CO2 + C COO-
H2CC
COO
OPO3
7.2
=GDPIDPADP
7.1
GTPITPATP
Mn2+ or Mg2+
+
O
-OOCCH2-
Addition of [14C]pyruvate does not give [14C]oxaloacetate.Pyruvate or enolpyruvate are not free intermediates.
(need large amount of enzyme so no nonenzymatic conversion of CO2 to HCO3-)
Scheme 7.2
In the absence of CO2, the enzyme acts like a kinase (H+ in place of CO2)
pyruvate
PEP carboxykinase-catalyzed reaction of PEP with ADP (no CO2)
C COO- + ATPPEP + ADP
7.3
CH3
O
Scheme 7.3
If the carboxylase reaction is run in D2O in the presence of malate DH/NADH, no D is in the malate;
oxaloacetate
Reduction of Oxaloacetate by Malate Dehydrogenase
CH COO-
OH
-OOCCH2C COO-
malate dehydrogenase
NADH7.2 7.4
O
-OOCCH2
malate
Malate dehydrogenase traps oxaloacetate to prevent nonenzymatic enolization.
therefore no enol of oxaloacetate formed.
Scheme 7.4
This mechanism is excluded by the previous result:
Hypothetical Mechanism for PEP Carboxykinase that Involves the Enolate of Oxaloacetate
H2C C
OPO3=
COO-
-OOC
O
COO- -OOC COO-
O
-OOC COO-
O
-OOC COO-
OC OO
HB PO3
= PO3=
-OOC
O
COO-
D
ADP ATP
7.5
D+
Scheme 7.5
Running the reaction in reverse
inversion ofstereochemistry
Stereochemistry of the Reaction Catalyzed by PEP Carboxykinase
O P O
O
O-
P
O
O-
O G
PS18O-
16O-
-OOCCOO-
O
O
COO-
PS 18O-
16O-
+ CO2 + GDP
7.6 7.7
+
Excludes covalent catalytic mechanism
Scheme 7.6
This Mechanism is Excluded:
Inconsistent with a double-inversion mechanism for PEP carboxykinase
-OOCCOO-
O
O
COO-
PO3=
O
C
O
O P
O
O-
O-
X-
X PO3=
GMP
GTP
Scheme 7.7
Possible Mechanism for PEP Carboxykinase
Concerted mechanism for PEP carboxykinase (or stepwise without release of intermediates)
OH2
O-
CO
O-P
O P GMP
O
O
O-PO
-OO
CCH2
O
C
O
OH2
O-
CO
O-P
O P GMP
O
O
O-PO
O-O
CCH2
O
C-O
O O-
O
O-
OH2
O-
CO
O-P
O P GMP
O
O
O-PO
-OO
CCH2
O
C
O
O O-
Mn2+ Mn2+Mn2+
Scheme 7.8
Same as PEP carboxykinase except Pi
instead of nucleotide diphosphateAll mechanistic experiments are the same for the two enzymes
Reaction Catalyzed by Phosphoenolpyruvate Carboxytransphosphorylase
PEP + CO2 + Pi
Mn2+ or Mg2+
OAA + PPi
(oxaloacetate)
Figure 7.2
re re
Alkene stereochemistry nomenclature rules for (Z)-1-bromo-1-propene (7.8)
C C
H
Br
H
CH3
C a
b
c
Cb
c
a
7.8
rere
Stereochemical Rules Needed to Determine Stereochemistry of PEP Carboxytransphosphorylase
Figure 7.3
si
re
Alkene Nomenclature Rules for (E)-1-bromo-1-propene (7.9)
C C
H
Br
CH3
H
C a
c
bCb
c
a
7.9
si 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
H 3H
CO2-
-O2C O
H 3H
CO2-
HO H
CO2-
3HH
CO2-
-O2C O
3HH
CO2-
OHH
CO2-
CO2-
OPO3=
H
3HH 3H
OPO3=-O2C
-O2C
H CO2-
H
-O2C
H CO2-
3H fumarase
malatedehydrogenase
3-S-[3H] OAA
(2re,3si)
(2S,3R) malate
_
malate
3 R-[3H]-OAA
7.10re-si
si-re
fumarase
(2si,3re)7.11-R
7.12-(2S,3R)
7.13-1H
+ 3HOH
H2O +
7.11-S
7.12-(2S,3S)7.13-3H
(2S,3S) malate
b
a dehydrogenase
-H2O
H
CO2
CO2
_H
Pi
Scheme 7.10
(Z)-[3-3H]PEP
With (E)-[3-3H]PEP, 98% 3H in fumarate; therefore carboxylation from si-re face
anti-elimination
anti-elimination
observed 98% loss as 3H2O
Two Possible Stereochemical Outcomes for Carboxylation of PEP Catalyzed by PEP Carboxytransphosphorylase
P-O bond of PEP breaks, but C-O bond of PEP breaks with EPSP synthase
fumarate
Scheme 7.11
blood-clotting proteins
binds Ca2+
Vitamin K Cycle for Carboxylation of ProteinsO
O
R
OH
OH
R
COOH
NHNH
O
COOH
NHNH
O
COOH
O
O
O
R
7.14
vitamin Kreductase
vitamin K carboxylaseCO2, O2
Gla
vitamin K epoxide reductase
R =
Glu
7.15
7.16
RSH
Figure 7.4
-proteases
Holds the proteases to the appropriate cells, triggering the blood-clotting cascade
Calcium-dependent Binding of Clotting Proteins to Cell Surfaces
NH
O
HN
O
COO
COO
NH
R
Ca2+
=
=
=
=
membrane bilayerGla
clotting proteins cell surface
O3PO
O3PO
O3PO
O3PO
Scheme 7.12
erythro- and threo-
erythro- F- elimination, but not threo-;
Test for Carbanion vs. Radical Mechanisms for Vitamin K Carboxylase
NH CO
FCOO-
NH CO
F
COO-
NH CO
NH
COO-
CO
F
COO-
NH CO
COO-
COO-F•
_
radical
carbanion
therefore stereospecific (carbanion)
Scheme 7.13
carboxylation with inversion of stereochemistry
Stereochemical Outcome of Vitamin K Carboxylase-catalyzed Carboxylation of
(2S,4R-fluoroglutamate)
carboxylaseNH
Glu Val
O
-OOC F
H
LeuPhe
H
vitamin K NHGlu Val
O
-OOC
F
COO-
LeuPhe
H
7.17
Scheme 7.14
But where does vitamin K fit into the mechanism?
Proposed Vitamin K Carboxylase-catalyzed Carboxylation of Glutamate Residues via a
Carbanionic Intermediate
H
C
HH
O O
O
C
OH
C
-OOC
O OO
O
+-
++
§ ***
§
:B
O- K+ O
O O-O
O
O
O
O
O
O
_
_
C
7.19
H
O
OEt
7.20
7.21
EtO2C
7.18
CO2Et
O2
Model Study for Function of Vitamin K
Not a strong enough base to deprotonate 7.20
Dieckmann condensation
Reaction does not work in absence of O2
Scheme 7.15
strong base
Base Strength Amplification Mechanism
Chemical model study for the activation of vitamin K1 as a base
Model for reducedvitamin K
Scheme 7.16
(not 1O2)
When run in 18O2, 0.95 mol atom 18O in epoxide 0.17 mol atom 18O in quinone oxygen
Two Proposed Mechanisms for Activation of Vitamin K1 as a Base
OH
R
OH
CH3
O
HO
R
O O-
CH3
O
HO OO
R
HO O-
O
OR
CH3CH3
O
R
OH
CH3
HO
R
OH
CH3
OO
OR
O
CH3
_
may pull offγ-protonfrom Gluresidues
7.22
OOH
H
R = phytyl
B-
OR
OCH3
R = phytyl
O +
O2A
BO2
-H++H+
HO-
-B
O O
O O
-B
O
O
OR
CH3
-HO-
To Determine Which Ketone is Involved
Incubation in 16O2 atmosphere gives loss of 0.17 mol atom 18O from 7.23, none from 7.24
O
18O
phytyl
CH3
18O
O
phytyl
CH3
7.247.23
Therefore, the ketone next to the methyl group is involved in the reaction
Scheme 7.18
To account for much loss of 18O from substrate
Modified Base Strength Amplification Mechanism for Vitamin K Carboxylase
O
18OH
phytyl
H Ophytyl
H18O O-O-
O-
phytyl
OH18O O
Ophytyl
H18O O-
O
Ophytyl
18O- OH
O
Ophytyl
O
O
Ophytyl
O
18O
weak base
strong base + HO-+ H18O-
7.26a 7.26b
O2
-S
Scheme 7.19
Bicarbonate as the Carboxylating Agent
PEPNo H2
18O formed(high enzyme concentration, short time at alkaline pH)
Reaction catalyzed by PEP carboxylase
CH2 C
O
COO-
P
O
O-
O-
-OOC CH2C
O
COO-+ HC18O3- + Pi(
18O)
1 18O atom2 (18O)
7.27
Mg++
Therefore HCO3-, not CO2
Scheme 7.20Note: nucleophilic mechanisms
concerted
stepwise associative
stepwise dissociative
No partial exchange detected ([14C]pyruvate does not give [14C]PEP)
Concerted (A), Stepwise Associative (B), and Stepwise Dissociative (C) Mechanisms for PEP Carboxylase
Therefore, either concerted or intermediate not released
HO
C
-OO -O
PO
O-
O
COO-
C
HO
-O O PO
O-
O-
O
COO-
-OO
COO-
18O
18
18 18
18
18
18Pi (
18O)18
A
HO
O O-
OP
-OO-
O
COO-
O-
COO
HO
OO
P
-O
O-
O -OO
COO-
18O
18
Pi (18O)
18
18
18
1818
18
Mg2+
B
HO
O O-
OP
-OO-
O
COO-
O
OO
P
-O
O-
O
18
1818
-OO
COO-
18O
18
O
C
O
18H
B:
18
18
O
COO
18
Mg2+
Pi (18O)
O
COOMg2+
18
C
Evidence for Stepwise Mechanism
in H218O inversion
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 concertedEvidence 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)
O
COOH
P
S17O
16O
7.29
P
S
17O
16O18O
7.28
Note: the ultimate carboxylating agent is CO2
Scheme not in text (after Scheme 7.20)
Mechanism for Incorporation of 18O into Substrate
Non-bridging 18O
HO
O O-
OP
-OO-
O
COO-
O
OO
P
-O
O-
O
18
1818
18
O
C
O
18H
B:
18
18
O
COO
Mg2+O
COOMg2+
18
-OP
-O
O-
O
18
B H
O
OO
P
-O
O-
O
18H
B:
18 18
O
COO
Mg2+
HO
O O-
OP
-OO-
O
COO-
18
18
1818
HO
O O-18
18
more than one 18Oincorporated into PEP
C
Biotin-dependent EnzymesMultisubunit enzymes
Enzyme reactions with HC18O3- give Pi with one 18O
and product with 2 18O atoms (bicarbonate)
Scheme 7.24
Covalent attachment of d-biotin to an active site lysine residue
HN NH
S
O
HH
H
(CH2)4COO-
HN NH
S
O
HH
HNH
O HN
O
HN
OS
H HH
NH-OOC
7.34 7.35
ATP AMP + PPi
142 o 118 o
7.34
Figure 7.5
Diagnostic method for biotin - add avidin KD = 1.3 10-15 M
Reactions Catalyzed by Biotin-dependent Carboxylases
C COO-
O
C SCoA
O
C SCoA
O
C COO-
O
C SCoA
O
CH3 CH
COO-
C
O
SCoA
O
SCoA
OCOO-
N
N N
N
NH2
OH OPO3=
O
ATP + HCO3- +
ATP + HCO3- +
P O
O
-OP
O
O-
ONH
O O
NH
SH
CH3H3C
HHO
ATP + HCO3- + + ADP + Pi
Pyruvate carboxylase
Acetyl CoA carboxylase
Propionyl CoA carboxylase
b-Methylcrotonyl CoA carboxylase
ATP + HCO3- +
+ ADP + Pi
+ ADP + Pi
+ ADP + Pi
H3C -OOCCH2
H3C -OOCCH2
CH3CH2 SCoA
CoASH
Scheme 7.25
Mechanism of Biotin-Dependent Carboxylases
No substrate or product needed
Suggests ATP activates bicarbonate
Partial exchange reaction of 32Pi into ATP (in absence of substrate) with biotin-dependent carboxylases
AT32P + Pi
ADPATP + 32Pi
M+2
HCO3-
Scheme 7.26
Mechanism for Partial Exchange of 32Pi into ATP with Biotin-dependent Carboxylases
O-HO
O
OPO3=HO
O
OPO3=HO
O
O-32PO3=HO
O-HPO4
3-
H32PO4=
O-HO
OADP
+ ATP + ADP
+ AT32P
Scheme 7.27
Partial Exchange Reaction of [14C]ADP into ATP with Biotin-dependent Carboxylases
[14C]-ATP + ADP[14C]-ADP + ATPHCO3
-/M2+
Scheme 7.28
[14C]product
substrate, HCO3-
ATP, M2+
[14C]substrate
Mechanism for Partial Exchange Reaction of [14C]ADP into ATP with Biotin-
dependent Carboxylases
[14C]ADP
C O
ATP
+ ADP
O
HO
[14C]ATP
PO3=HCO3
-
(reaction is reversible)
Scheme 7.29
Evidence for Enzyme-Bound IntermediateIn the absence of pyruvate get a carboxylated enzyme
if pyruvateis added
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)
Pyruvate carboxylase-catalyzed incorporation of 14C from H14CO3
- into the enzyme
C
O
XO-HO
O
HO
C
O
XHO
O
C COOHH2C
O
C COOH
+ Pi
CH2-X
+ ATP
HOO14C
+ ADP
+ +
14
H
14
:B
14
-X
M2+
NHN
S
O
CH3OC
O
NH(CH2)4
O
CHCOO-
NH3+
NHN
S
O
CH3OC
O
COO-
biotinidase
Lys +
NHN
S
O
CH3OC
O
NH(CH2)4
O
CH
HN
CO
NH
CO
trypsin
papain
7.36
Scheme 7.30
Isolated; X-ray crystal structure
The X in previous Scheme
Isolation of N1-methoxycarbonylbiotin from the Reaction Catalyzed by Pyruvate Carboxylase Followed
by Diazomethane Trapping of the N-carboxybiotin
Figure 7.61.
2.
3.
Six Possible Mechanisms for Formation of N1-carboxybiotinO
HOC OPO3=
NHN
S
O
O
C OPO3=
NHN
S
O
R
NHN
S
O
C
O
HO
RR
NHN
S
O=O3P
R
O
C-OOH
NHN
S
O=O3P
R
NHN
S
O-HO
R
OPO3=
O
O
H
O
C
O
H
B
NHN
S
O
H
R
B:NHN
S
O
R
+ ADP•Mg2+
7.37
7.37
=O3P O
HCO3- + ATP•Mg2+
P O P OAdo
stepwiseNHN
S
O
H
R
B:NHN
S
O
R
7.37
=O3P O P O P OAdo
O
O-
O
O-
-ADP
PO43-
O O
O-O-
PO43-
-ADP
NHN
S
O
R
OOH
O
PO3=
-PO44-
CO2
HCO3-
Mg2+
Mg2+
Figure 7.6
4.
5.
6.
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).
NHN
S
O
R
HOC
O-O
BH
NHN
S
O
C
R
OH
HO
O-
AdoO P O P OPO3
=
NHN
S
O
R
O-
HO
O=O3P
NHN
S
O
R
O
COHO
NHN
S
O
C
R
O
O
OH
AdoO P O P OPO3
=
=O3P
NHN
S
O
H
R
B:
NHN
S
O
H
R
B:
NHN
S
O
H
R
B:
=O3P O P O P OAdo
NHN
S
O
R
PO
-O
O-
OH
COO
BH
NHN
S
O
R
P
OH-O
-OOO
OH NH:N
S
O
R
PHO
O-
O-
ADP
ADP
7.37
O
Pi
O
OH
ADP
O- O-
OO
O- O-
OO
O
O- O-
O
7.37
Pi
7.37
Pipseudorotation
-H+
Mg2+
Mg2+
Mg2+
Mg2+
Mg2+
Scheme 7.31
carboxyphosphate
Mechanism for the Formation of Carboxyphosphate in the Reaction
Catalyzed by Acetyl-CoA Carboxylase
H18O 18OPO3
=
18O
P O P O P OAdo + ADP
[18O] Pi + C18O2
O
O-
-O
O
O-
O
O-
HC18O3-
H2O
Figure 7.8
Initial evidence for concerted: retention of configuration at -carbon
Possible Mechanisms for Transfer of CO2 from N1-carboxybiotin to Substrates
B Stepwise-associative
NHN
O
-OOC
-OOC CH2
HO
-OOC CH2
COO-
O
-OOC CH2H
O - B
NHN
O
-OOC
-OOC CH2-OOC
O
CH2
COO-
O
-OOC CH2
H
O
-OOC CH2
C
O
OONHN
O
C
-OOC CH2
COO-
O
S R
_
S
associative
R
_
S
A Concerted
R
dissociativeO
O
C Stepwise-dissociative
Scheme 7.37
Evidence for Stepwise Mechanism
Double isotope fractionation test:
Compare with 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 2H13(V/K) for 13CH3COCOOH 1.022713(V/K) for 13CD3COCOOH 1.0141 (calculated value is 1.0136)
therefore stepwise
Transcarboxylase and propionyl-CoA carboxylase-catalyzed elimination of HF from -fluoropropionyl-CoA
13CH3COOH
O
13CD3COOH
O
CoASCoAS
B-
F
O
H
O
+ HF
7.437.44