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