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The sulfhydryl groups of citrate-condensing enzyme

The sulfhydryl groups of citrate-condensing enzyme

Vol. 18, No. 1, 1965 8tOCHEMlCAL AND 8lOPHYSICAL RESEARCHCOMMUNICATIONS THE SULFHYDRYL GROUPS OF CITRATE-CONDENSING ENZYME* Paul A. Srere Lawrenc...

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Vol. 18, No. 1, 1965

8tOCHEMlCAL AND 8lOPHYSICAL RESEARCHCOMMUNICATIONS

THE SULFHYDRYL

GROUPS OF CITRATE-CONDENSING

ENZYME*

Paul A. Srere Lawrence

Radiation

Laboratory,

Livermore,

University

of California

California

Received October 23, 1964 Until

recently

it has been generally

(SH) group is not involved enzyme. is fully

in the active

Stern (1960) has reported active

after

treatment

assumed

that a sulfhydryl

site of citrate-condensing

that citrate-condensing

with 10

-3

enzyme

-M p-chlormercuribenzoate

(pCMB).

We have observed

(Broder

et al.,

densing

enzyme of pig heart

contains

SH groups that react

solution

with pCMB ,with no great reduction

also reported

(Brazil

and Srere,

known to react with SH groups In addition,

we routinely

1963) in the presence

activity.

We have concluded

participate

an SH reagent,

in the enzymatic

Eggerer

do not inhibit

of 5, 5 I-dithiobis

1959).

et al,

Although treatment maleimide

activity.

compounds

which are

of the enzyme

(Srere et.,

(2-nitrobenzoate)

(DTNB)

effect

on enzyme

that the SH groups on the protein

do not

reaction.

enzyme

is essential

which involves

and his co-workers

of the enzyme

We

of the enzyme.

(1964) on the other hand have reported

a mechanism

Eggerer

in aqueous

the activity

with no apparent

SH group of citrate-condensing have proposed

of enzymatic

1963) that other

assay the activity

(Ellman,

1962) that the citrate-con-

with either

does not cause an inhibition

Work done under the auspices sion.

for its activity

it in the catalytic

the observations

iodoacetamide

or N-ethyl

of activity,

and

reaction.

confirmed

of the U. S. Atomic

87

that the

that

they report

that

Energy

Commis-

Vol. 18, No. 1,1965

BIOCHEMICALAND BIOPHYSICALRESEARCHCOMMUNICATIONS

1.3 x 1o-3 -M pCMB incubated resulted

in loss of enzyme

sults (Srere, enzyme

1963),

results

I have, a low molar activity

with the enzyme for 30 minutes

activity.

Further,

which show that acetylation

in loss of activity, in addition,

ratio

they interpret

as indicating

unpublished

of iodine to enzyme

is observed,

In view of the possibility

an involvement which

a complete

inhibition

that the SH groups

our re-

of citrate-condensing

experiments

which may also be evidence

at 0”

of SH.

show that at of enzyme

for SH involvement.

involved

in the reaction

might be unavailable

to certain

SH reagents,

ancies

of Eggerer

and others,

we have carried

understand

the role of the SH groups

in the results

experiments

to attempt

to better

together

with the discrepout more

in the reaction. Amino

acid analysis

indicates

l/ 2 cystine

per mol of enzyme.

in aqueous

solution

(Table 1); however,

Table

Amino

sulfhydryl

to react

can be detected

with DTNB plus urea 4 mols of SH can be titrated.

groups

procedure

acid analysisa

of about 6 mols of

When the enzyme is allowed

with DTNB no free

1. The sulfhydryl

Analytical

the presence

(l/2

of citrate-condensing

Mols

enzyme.

SH/ 80,000 g ‘proteinb 6

cystine)

0

DTNB in 0.1-M

tris

pH 8.1

DTNB in 0.1-M 4.0-M

tris pH 8.1 urea

3.8

pCMB in 0.1-M

tris

pH 8.1

l.gc

pCMB in 0.1-M 4.0-M

tris pH 8.1 urea

4.4

aKindly performed by Dr. L. Levintow. b Our recent studies on the sedimentation coefficient.of the enzyme would indicate that this figure is a better value for the molecular weight of the enzyme than earlier figures. ‘This value is obtained in 5 minutes before precipitation of the No precipitation occurs with pCMB in urea. enzyme occurs. 88

BlOCHEMlCAL AND BIOPHYSICALRESEARCHCOMMUNlCAliONS

Vol. 18, No. 1, 1965

Their

rate of appearance

depends upon the urea concentration

first-order

for over 90% of reaction

tion pCMB

shows the presence

but further

titration

precipitate. be titrated

(Srere,

of 2 mols

is obscured

because

With pCMB plus urea, and no precipitation

1964).

In aqueous

of SH immediately the pCMB

however,

occurs

and is solu-

(Fig.

enzyme begins to

4 mols of SW groups

(Table

l),

1 and Fig.

can

1).

0.100

MINUTES

Fig. 1. The reaction of pCMB with citrate-condensing enzyme. Each cuvette contained 100 pmols tris-HCl, pH 8.1, and 200 fig (2.5 rrqumols) citrate-condensing enzyme in a total volume of 1.0 ml. The An reaction was started by the addition of 10 ~1 of 5 X 10V3-M pCMB. appropriate control without enzyme was run simultaneously.

If one looks at the activity (Table 2), there

is no correlation

When half of them are covered mains; little

when 94% of the activity change has occurred

has no sulfhydryl or pCMB-enzyme allowed

tent is seen,

between with pCMB,

(or N-ethyl

periods

and activity.

with acetic

enzyme,

of time,

Hg-enzyme If Hg-enzyme

not shown here)

then precipitation

10 -3 -M iodoacetamide

any observable

cannot be assayed for activity 89

loss of activity.

is

occurs

does not react

(15 min at 0” ) since no change in the enzyme

nor is there

re-

anhydride,

of the enzyme.

46 % of its activity.

maleimide

proteins

then 7% of the activity

is inhibited

and retains

modified

SH groups

in the SH content

can be detected.

with the enzyme

enzyme

groups

to stand for longer

and no activity

of the variously

SH con-

DTNB-

since it can only be formed

in

Vol. 18, No. 1,1965

8lOCHEMlCAl AND 8lOPHYSlCAL RESEARCHCOMMUNICATIONS

urea and enzyme ent (Srere,

activity

is lost in urea whether

or not DTNB is pres-

1964). Table 2.

Relation

Preparation

of SH groups to enzyme % activity

Enzyme

activity.

Mols SH/80,000

100

3.ga

6

3.6a

Acetyl-enzyme Mercuri-enzymeb

46

OC

pMB- enzyme

7gd

me

g protein

aMeasured with DTNB in 6.8-M urea. b5.0 x 10-4 -M protein SH incubated 15 min at 25’ with 5.0 X 10s4-M HgC12. ‘Measured with pCMB with and without 4-M urea. dThis is 15 minutes after addition of pCMB and over half of the SH groups have been titrated; precipitation had proceeded (as judged by turbidity at 320 w) for 2-3 minutes. eBy difference.

Several vestigations

other observations are pertinent

tions has varied protein;

from

whereas

to this question.

the corresponding

with SH compounds

enzyme

the existence

teins in a preparation

activities

of one form

to the other

conversion

that did not affect the enzyme

varied

from

We have

glutathione).

of two electrophoretically

conversion

of prepara-

has ever been seen

cysteine,

which is homogeneous

in-

to 4 mols per mol of

of preparations

(mercaptoethanol,

of other

The SH content

1 mol per mol of protein

about 70 to 1000/o. No activation

reported

made by us in the course

separable

active

pro-

in the ultracentrifuge.

The.

seemed to depend upon an SH to SS activity

(Broder

and Srere,

1963). The available

data are consistent

of citrate-condensing bonding enzyme.

to maintain

enzyme the proper

It is unlikely

with the idea that the SH groups

are all involved conformation

that a subunit 90

structure

in internal of the active is involved

hydrogen site of the since no

Vol. 18, No. 1, 1965

appreciable

8IOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS

change was observed

urea or sodium

dodecyl

in the sedimentation

of enzyme

in

sulfate.

REFERENCES Brazil. H., Broder, I., 252 (1962). Broder, (1963).

I.,

Eggerer, H., 436 (1964).

and Srere, Bhaduri,

P. A., A.,

and Srere, Remberger,

Federation

and Srere,

P. A., U.,

Proc.

P. A.,

Biochim.

22, 240 (1963).

Federation

Biophys.

and Griinewalder,

Proc.

Acta, C.,

67, 626

Biochem.

Ellman, G. , Arch. Biochem. Biophys. 62, 70 (1959). Srere, P. A., Biochim. Biophys. Acta, 77, 693 (1963). Srere, P. A., Brazil, H., and Gonen, L., Acta Chem. Stand., S129 (1963). Srere, P. A., unpublished observations (1964). Stern, J. R., and Myrbick, 377 (1961).

The Enzymes, 2nd Edition, Boyer, K., editors, Volume 5. Academic

91

21,

Z, 339,

17,

P. D., Lardy, H., Press, New York, p.