Vol. 73, No. 3, 1976

BIOCHEMICAL

CARBON

ISOTOPE

AND BIOPHYSICAL

EFFECT

ON THE

DECARBOXYLATION

OF

Marion Department

Received

August

of

H.

RESEARCH COMMUNICATIONS

ENZYMATIC

PYRUVIC

ACID

O’Leary

Chemistry, University Madison, Wisconsin

of

Wisconsin

53706

24,1976

The decarboxylation of pyruvic acid by the thiamine pyrophosphate dependent pyruvate decarboxylase from brewer’s yeast is accompanied by a carboxyl carbon isotope effect k12/k’3 = 1.0083+0.0003 at 25O, pH 6.8. The small size of the isotope effect indicates that decarboxylation is not rate-determining in the overall reaction. The rate constant for decarboxylation of the enzymebound pyruvate-thiamine pyrophosphate complex is greater by about a factor of five than the rate constant for dissociation of this complex to form free pyruvate and the enzyme-thiamine pyrophosphate complex.

INTRODUCTION Carboxyl to

carbon

estimate

complex

the

rate

of

to

the

relative

plex

to

isotope

regenerate

effects

on

decarboxylation rate

free

of

enzymatic of

the

dissociation

enzyme

and

free

decarboxylations appropriate

of

the

substrate

can

be

used

enzyme-substrate

substrate (1,2).

For

from

this

com-

the

simple

mechanism kl

k3

E+Se

ES

-

EP

+

for

the

CO2

-+

E

+

P

+

CO

(1)

2

k2 in

kl

which

the

enzyme-substrate

and

k

bon

isotope

3

is

k2 are

and

the

rate complex

rate effect

-

for

is

by

given

=

k3

k13

tope eq.

Copyright All rights

that effects

1 may

be

kl

and

a single

decarboxylation,

and the

the

+

k3/k2

1

+

k3/k2

dissociation

of

decarboxylation observed

step

carboxyl

car-

(2)

is k2.

formation

preceding

‘*/k313

decarboxylation on

not

immediately

constant

kl* observed

provided

constants

The step.

0 I976 by Academic Press, Inc. of reproduction in any form reserved.

irreversible reaction In

and

that

represented

such

614

a case,

there by

eq.

2

rate is

are no carbon kl

constant replaced

by

a

isoin

Vol.

73,

similar

but

more

this

entirely

from

functions

by

effects

it

AND

is

BIOPHYSICAL

which

the

of

enzymatic

usually

RESEARCH

ratio

k

3/k 2

COMMUNICATIONS

is

replaced

decarboxylations

found

that

by

a

have

decarboxylation

been is

(2). dependent

the to

pyrophosphate

MATERIALS

and

pyrophosphate

isotope

thiamine

in

A variety

(1).

technique,

thiamine

AND

equation

rate-determining

yeast

carbon

complex

function

by

The

BIOCHEMICAL

more

complex

studied not

3,1976

No.

mechanism study

the

pyruvate shown

fate

of

in

decarboxylase Scheme

the

I

(EC

(3,4).

covalent

4.1.1.1)

We have

used

enzyme-pyruvate-

complex.

METHODS

All materials were of reagent grade and were shown not to ties which would interfere with the isotope effect measurements. were conducted on a Gilford Model 222 spectrophotometer at 25’. ratios were measured on a Nuclide Associates RMS 6-60 isotope-ratio t rometer .

contain impuriEnzyme assays Isotope mass spec-

Pyruvate decarboxylase was purified to homogeneity from brewer’s yeast by During the course of purification the enzyme was the method of Ullrich (5). assayed by observing the disappearance of NADH on reduction of acetaldehyde by alcohol dehydrogenase. For the isotope effect experiments the enzyme was assayed spectrophotometrically at 313 nm (A& = 20). Carbon isotope effects were determined by isotope ratio measurements of two parallel samples of carbon dioxide obtained from the decarboxylation of pyruvic acid (6). A solution of 0.03 M sodium pyruvate in 0.05 M phosphate buffer, pH 6.80, containing 1 mM Mg2+ and 10 uM thiamine pyrophosphate was divided into two parts and each was freed of CO2 by purging with C02-free nitrogen for I hr. Following equilibration at 25O, known amounts of pyruvate decarboxylase which had been desalted in COz-free buffer were added to both solutions. One solution was allowed to decarboxylate to the extent of approximately 10% and then the reaction was stopped by addition of concentrated H2SOs. The other sample was allowed to decarboxylate completely before addition of acid. Completeness of decarboxylation in the latter sample was checked by neutralization of the solution and assay for pyruvate with lactic dehydrogenase after the CO2 had been removed. The CO2 liberated from both solutions on acidification was removed and repeatedly distilled on the vacuum line. The CO2 samples following this procedure were free of Hz0 and acetaldehyde. Isotope ratios and isotope effects were calculated as described previously (6). RESULTS Results effect All ratios error,

of

on

the

as

independent

enzymatic

experiments for

six

all expected.

measurements

decarboxylation

were

conducted

100%

decarboxylation The

carbon

of of

with

the

same

samples isotope

effect

615

the

carboxyl

pyruvic

acid

lot

of

are

are

k

12

in

so

the

within

/k

13

isotope

shown

pyruvate,

identical is

carbon

Table isotope

experimental

= 1.0083+0.0003.

I.

Vol.

73, No.

TABLE

3,1976

I.

BIOCHEMICAL

Carboxyl carbon pyruvic acid at 1.0 mM Mg2+ and

AND

isotope effects 250 pH 6.8, Id pM thiamine

Percent react ion

Isotope

BIOPHYSICAL

in

RESEARCH

on the enzymatic 0.05 M phosphate pyrophosphate.

ratiosa

x

COMMUNICATIONS

decarboxylation buffer containing

of

IO6 k12/k’3

low conversion

100% conversion

11

14432

14539

1.0083

11

14439

14546

1.0083

11

14437

14544

1.0083

14447

14562

1.0086

IO

14445

14547

1.0078

11

14438

14549

1.0086

5

Mean

1.0083 +o .0003

aMass spectrometer reference standard,

ratios but

m/e not

46/44 corrected

corrected for the

to a constant presence of

value,of oxygen-17.

a CO2

DISCUSSION A small

but

decarboxylation observed acid

of in

(7),

significant

and

the

carbon

pyruvic

acid

enzymatic arginine

observed

in

nonenzymatic

entirely

rate-determining

observed

in

somewhat

faster

effect The

pH 6.8.

decarboxylation *

.

The

isotope

of effect

(2).

in

Small

isotope

reactions preceding

steps

in in

is

which the

observed

effect

is

glutamic is

decarboxylations

decarboxylation than

at

isotope

in

the

enzymatic

smaller

than

those

(6),

acetoacetic

acid

considerably

which

the

smaller decarboxylation

effects

such

as

the

decarboxylation

reaction

mechanism

those

step

this

one step

are is

(2).

12 13 = 1.0144+0.0004 has recently been *A carbon isotope effect k /k for the enzymatic decarboxylation of arginine by bacterial argiriine lase at 25’, pti5.25, by O’Leary and Piazza (unpublished results).

616

than

obtained decarboxy-

is

BIOCHEMICAL

Vol. 73, No. 3,1976

Scheme

The

carbon

interpreted

of

kg/k,,

thiamine vate

effect

on

the

mechanism

effect

and is

steps the

and

ratio

Based

isotope

on

effect

This

rate

time

the

range

step

us

in

step

the is

complex usually complex

of

the

to

of

its

carbon

isotope

studies,

it

faster the

in

is,

another

decarboxylation, pyruvate

and

617

expected

its the

covalent

but thiamine

this perhaps

pyrophosphate.

2 of

to

esti-

pyruvatefree

pyruof

the

decarboxylation that

a carbon

for

this 5-7

step

not

the the

state

(2).

k3/k2.

for

Instead,

transition

way,

eq.

used

is

acid.

2.

be made

approximately

decarboxylation

which

Stated

argued

eq.

to

the

be

the

covalent

can on

been

pyruvic

(that

step

of

that of

estimate

is

a value

be

the

effect

can

in

decomposition

an

has

by

taken

2 can of

that

indicates

is

Eq.

rate

calculate

formed.

undergoes reverts

the

given

account

‘2 /k ‘3 = 1.04-1.06

slightly

is

no

acid between

I is

decarboxylation

decarboxylation

than

Scheme

pyruvic

relationship

decarboxylation.

model

to

of

of

provided

k

decarboxylase.

The

(8),

to

ratio

energy)

pyrophosphate formed,

the

constant

lower

once

of

in

carboxylation slightly

results

pyruvate

I.

Scheme

rate

k312/k313,

enables

determining

the

RESEARCH COMMUNICATIONS

decarboxylation

irreversible the

of

of

mechanism

pyrophosphate, of

estimate

the

complex

thiamine value

step.

of

following

pyrophosphate

expected

This

action

decarboxylation

rates

mate

of

the

isotope

Because

Mechanism

isotope

using

observed

the

I.

AND BIOPHYSICAL

ratede-

has

pyruvate-thiamine covalent lo-20%

complex, of It

the should

a

Vol.

be

73, No. 3,1976

noted

that

explain It

this

the

observed

has

been

enzymatic the

BIOCHEMICAL

reversion

must

isotope that

decarboxylation

of complex

effect

experiments

proposa

I.

t

is

BIOPHYSICAL

proceed

all

the

RESEARCH

way

to

free

COMMUNICATIONS

pyruvate

to

effect.

suggested

enzyme-product

AND

the pyruvic

(g), not

overall

rate

acid

is

Because possible

of to

the the

evaluate

determining

step

release nature

of of

the

in

the

acetaldehyde the

carbon

correctness

from isotope

of

this

ACKNOWLEDGMENTS We are

gratefu

yeast.

This

research

Science

Foundation.

to was A.

the

Miller

Brewing

supported Miller

provided

by

Company

grant

for

PCM75-15315

experimental

supplying from

brewer’s the

National

assistance.

REFERENCES I. 2. 2:

2: i: 9.

O’Leary, M. H. (1877) in “Transition States of Biochemical Processes”, R. L. Schowen and R. Gandour, eds., Plenum Pub1 ishing Corp., New York. O’Leary, M. H. (1976) Bioorg. Chem., in press. Krampitz, L. 0. (1863) Ann. Rev. Biochem. 38, 213-240. Ullrich, J., Ostrovsky, Y. M., Eyzaguirre, J., and Holzer, H. (1970) Vitamins and Hormones 28, 365-388. Ullrich, J. (1970) Methods in Enzymology 18A, 109-115. O’Leary, M. H., Richards, D. T., and Hendrickson, D. W. (1970) J. Am. Chem. Sot. 92, 4435-4440. R. L. (1972) J. Am. Chem. Sot. 94, 626-630. O’Leary, M. H., and Baughn, Burton, K., and Krebs, H. A. (1853) Biochem. J. 54, 94-l 07. and Schellenberger, A. (1970) Z. Phyxol. Chem. 351, 1435-1440. Hiibner, G.,

618