The Organic Chemistry of EnzymeCatalyzed Reactions Chapter 7 Carboxylations

1
The Organic Chemistry of Enzyme-Catalyzed
Reactions Chapter 7 Carboxylations
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CarboxylationsGeneral 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

3

• 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
  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)

4
Test for whether CO2 or HCO3- is the substrate
for a carboxylate
Carboxylations
CO2 H2O H2CO3
(equilibrium 1 min)
electrophile
nucleophile
Possible outcomes when CO2 is added to a
carboxylase
Figure 7.1
Also, repeat in the presence of carbonic
anhydrase (catalyzes hydrolysis of CO2 ? H2CO3)
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Reaction catalyzed by PEP carboxykinase
CO2 as Carboxylating Agent
Scheme 7.1
oxaloacetate
PEP
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 14Cpyruvate does not give
14Coxaloacetate.
Pyruvate or enolpyruvate are not free
intermediates.
6
PEP carboxykinase-catalyzed reaction of PEP with
ADP (no CO2)
In the absence of CO2, the enzyme acts like a
kinase (H in place of CO2)
pyruvate
Scheme 7.2
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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.
oxaloacetate
malate
Scheme 7.3
Malate dehydrogenase traps oxaloacetate to
prevent nonenzymatic enolization.
8
Hypothetical Mechanism for PEP Carboxykinase that
Involves the Enolate of Oxaloacetate
This mechanism is excluded by the previous result
Scheme 7.4
9
Stereochemistry of the Reaction Catalyzed by PEP
Carboxykinase
Running the reaction in reverse
Scheme 7.5
inversion of stereochemistry
Excludes covalent catalytic mechanism
10
Inconsistent with a double-inversion mechanism
for PEP carboxykinase
This Mechanism is Excluded
Scheme 7.6
11
Concerted mechanism for PEP carboxykinase (or
stepwise without release of intermediates)
Possible Mechanism for PEP Carboxykinase
Scheme 7.7
12
Reaction Catalyzed by Phosphoenolpyruvate
Carboxytransphosphorylase
(oxaloacetate)
Scheme 7.8
Same as PEP carboxykinase except Pi instead of
nucleotide diphosphate All mechanistic
experiments are the same for the two enzymes
13
Alkene stereochemistry nomenclature rules for
(Z)-1-bromo-1-propene (7.8)
Stereochemical Rules Needed to Determine
Stereochemistry of PEP Carboxytransphosphorylase
re
re
Figure 7.2
14
Alkene Nomenclature Rules for (E)-1-bromo-1-propen
e (7.9)
si
Figure 7.3
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
15
Two Possible Stereochemical Outcomes for
Carboxylation of PEP Catalyzed by PEP
Carboxytransphosphorylase
(Z)-3-3HPEP
anti-elimination
With (E)-3-3HPEP, 98 3H in fumarate therefore
carboxylation from si-re face
anti-elimination
fumarate
observed 98 loss as 3H2O
P-O bond of PEP breaks, but C-O bond of PEP
breaks with EPSP synthase
Scheme 7.10
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Vitamin K Cycle for Carboxylation of Proteins
blood-clotting proteins
binds Ca2
Scheme 7.11
17
Calcium-dependent Binding of Clotting Proteins to
Cell Surfaces
Figure 7.4
-proteases
Holds the proteases to the appropriate cells,
triggering the blood-clotting cascade
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Test for Carbanion vs. Radical Mechanisms for
Vitamin K Carboxylase
Scheme 7.12
erythro- and threo-
erythro- F- elimination, but not threo-
therefore stereospecific (carbanion)
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Stereochemical Outcome of Vitamin K
Carboxylase-catalyzed Carboxylation of
(2S,4R-fluoroglutamate)
carboxylation with inversion of stereochemistry
Scheme 7.13
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Proposed Vitamin K Carboxylase-catalyzed
Carboxylation of Glutamate Residues via a
Carbanionic Intermediate
Scheme 7.14
But where does vitamin K fit into the mechanism?
21
Chemical model study for the activation of
vitamin K1 as a base
Model Study for Function of Vitamin K
Not a strong enough base to deprotonate 7.20
Model for reduced vitamin K
Reaction does not work in absence of O2
strong base
Dieckmann condensation
Scheme 7.15
Base Strength Amplification Mechanism
22
Two Proposed Mechanisms for Activation of Vitamin
K1 as a Base
(not 1O2)
Scheme 7.16
When run in 18O2, 0.95 mol atom 18O in epoxide
0.17 mol atom 18O in quinone oxygen
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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
Therefore, the ketone next to the methyl group
is involved in the reaction
24
Modified Base Strength Amplification Mechanism
for Vitamin K Carboxylase
To account for much loss of 18O from substrate
Scheme 7.18
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Reaction catalyzed by PEP carboxylase
Bicarbonate as the Carboxylating Agent
PEP
No H218O formed (high enzyme concentration, short
time at alkaline pH)
Scheme 7.19
Therefore HCO3-, not CO2
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Concerted (A), Stepwise Associative (B), and
Stepwise Dissociative (C) Mechanisms for PEP
Carboxylase
Note nucleophilic mechanisms
Scheme 7.20
concerted
stepwise associative
stepwise dissociative
No partial exchange detected (14Cpyruvate does
not give 14CPEP)
Therefore, either concerted or intermediate not
released
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Evidence for Stepwise Mechanism
inversion
in H218O
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)
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Mechanism for Incorporation of 18O into Substrate
C
Non-bridging 18O
Scheme not in text (after Scheme 7.20)
Note the ultimate carboxylating agent is CO2
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Covalent attachment of d-biotin to an active site
lysine residue
Biotin-dependent Enzymes Multisubunit enzymes
Scheme 7.24
Enzyme reactions with HC18O3- give Pi with one
18O and product with 2 18O atoms (bicarbonate)
30
Reactions Catalyzed by Biotin-dependent
Carboxylases
Figure 7.5
Diagnostic method for biotin - add avidin
KD 1.3 ? 10-15 M
31
Partial exchange reaction of 32Pi into ATP (in
absence of substrate) with biotin-dependent
carboxylases
Mechanism of Biotin-Dependent Carboxylases
Scheme 7.25
No substrate or product needed Suggests ATP
activates bicarbonate
32
Mechanism for Partial Exchange of 32Pi into ATP
with Biotin-dependent Carboxylases
Scheme 7.26
33
Partial Exchange Reaction of 14CADP into ATP
with Biotin-dependent Carboxylases
Scheme 7.27
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Mechanism for Partial Exchange Reaction of
14CADP into ATP with Biotin-dependent
Carboxylases
Scheme 7.28
(reaction is reversible)
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Pyruvate carboxylase-catalyzed incorporation of
14C from H14CO3- into the enzyme
Evidence for Enzyme-Bound Intermediate In the
absence of pyruvate get a carboxylated enzyme
Scheme 7.29
if pyruvate is 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)
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Isolation of N1-methoxycarbonylbiotin from the
Reaction Catalyzed by Pyruvate Carboxylase
Followed by Diazomethane Trapping of the
N-carboxybiotin
Scheme 7.30
The X in previous Scheme
Isolated X-ray crystal structure
37
Six Possible Mechanisms for Formation of
N1-carboxybiotin
1.
Figure 7.6
2.
3.
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4.
5.
6.
Figure 7.6
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).
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Mechanism for the Formation of Carboxyphosphate
in the Reaction Catalyzed by Acetyl-CoA
Carboxylase
carboxyphosphate
Scheme 7.31
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Possible Mechanisms for Transfer of CO2 from
N1-carboxybiotin to Substrates
Figure 7.8
Initial evidence for concerted retention of
configuration at ?-carbon
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Transcarboxylase and propionyl-CoA
carboxylase-catalyzed elimination of HF from
?-fluoropropionyl-CoA
Evidence for Stepwise Mechanism
Scheme 7.37
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 2H 13(V/K) for 13CH3COCOOH
1.0227 13(V/K) for 13CD3COCOOH 1.0141
(calculated value is 1.0136)
therefore stepwise