Phenol-sulfotransferase inactivation by 2,3-butanedione and phenylglyoxal: Evidence for an active site arginyl residue

BIOCHEMICAL

Vol. 78, No. 3, 1977

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PHENOL-SULFOTRANSFERASE INACTIVATION BY 2,3-BUTANEDIONE AND PHENYLGLYOXAL: EVIDENCE FOR AN ACTIVE SITE ARGINYL RESIDUE* Ronald

T.

Borchardt

and Charles

S. Schasteen

Department of Biochemistry, McCollum Laboratories, University of Kansas, Lawrence, Kansas 66044 Received

August

22,1977

SUMMARY: In an attempt to determine if arginyl residues play a role in sulfate transfer reactions, we studied the effects of 2,x-butanedione and phenylglyoxal, both chemical modifying agents for arginyl residues, on phenol-sulfotransferase. Both reagents produced rapid inactivation of the enzyme, with the inactivation following pseudo-first order kinetics. The rate of inactivation was dependent upon the concentration of the chemical modifier. Competition studies showed that inclusion of 3'-phosphoadenosine5'-phosphosulfate during the preincubation step protected the enzyme from inactivation. The results suggest a possible role for arginyl residues as anionic recognition sites for sulfate transfer reactions. Riordan anion fer

recognition

reactions

catalyzed which

well

(EC 2.8.2.1).

by the

they

play

reaction,

enzyme phenol-sulfoan important

group

3'-phosphoadenosine-5'-

residues a role

exist

at the

as anionic

of PAPS, we have studied

the

(2-5).

effects

PST catalyzes

substrate. active

recognition of

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

as

(e.g.

etc.)

acceptor

in

compounds,

To desite

sites

of

PST

for

the

2,3-butanedione

*The abbreviations used are: PST, phenol-sulfotransferase; PAPS, 3'-phosphoadenosine-5'-phosphosulfate.

Copyright All rights

role

neurotransmitters

from

trans-

enzyme

serotonin,

(PAPS) to a phenolic arginyl

transfer

phenol-containing

of endogenous

of a sulfate

whether

and whether

of a sulfate

are

phosphate

An analogous

PST plays

of xenobiotic

residues

catalyze

pathway.

represented

metabolism

phosphosulfate

binding

glycolytic

is

arginyl

on enzymes which

norepinephrine,

transfer

termine

have shown that

would be that

(PST)

detoxification

dopamine, the

in the

in our study

as the

(1) sites

process

transferase the

et al. --

BIOCHEMICAL

Vol. 78, No. 3, 1977

and phenylglyoxal,

both

chemical

residues

(6,7),

activate

PST and evidence

an arginyl sulfate

donor

gr)

from rat

according

with

of protein/min

using

Ci/mmole)

described

transfer

of

tation

tillation

of

on PST for

the

of the

dione

(Aldrich

were counted

Nuclear,

following of

0.4

components

ml:

Chemical

in 50 mM borate Phenylglyoxal volume

(Sigma)

experiment

Co.)

(variable

buffer,

is based on the then

using

the

(0.5-

by liquid

scin-

appropriate

using

the

was performed

by

en-

(11).

this

sequence

pH 7.40;

concentration)

conin a

2,3-butanewhich

was

and the enzyme prepara-

experiments

were carr?ed

125 mM bicarbonate

1068

precipi-

2,3-butanedione

buffer,

pH 7.40;

inactivation

of 0.4 ml using

with

added in

50 mM borate

1.02-

of a previous-

PAPS was prepared

and purification

inactivation

per

formed/mg

Aliquots

were corrected

Unlabeled

protein

and ZnSOq, leaving

in solution.

The results

step

Enzyme activ-

The assay

Ba(OH);,

on Ecteola-cellulose

A typical

volume

(New England

(9).

supernatant

(8).

0.1 pmol product

by a modification

assay

method of Roy (10)

total

42 mg of

as a substrate.

[35 S]PAPS with

blanks.

chromatography

a total

for

procedure

35 SO4 from [35 S]PAPS to p-nitrophenol,

p-nitrophenol

tion.

activity

[ 35 SIPAPS

radiochemical

counting.

prepared

a role

(NH4)2S04 fractionation

contained

and p-nitrophenol

1 ml) of the resulting

sisted

in-

Sprague-Dawley,

reported

which

p-nitrophenyl[35S]sulfate

zymatic

support site

(male,

the

p-nitrophenol

using

of unreacted

product

through

a specific

was determined

liver

to a previously

in a preparation

milliliter

ly

were shown to

here to

recognition

arginyl

and Methods

resulting

2.03

is provided

for

PAPS.

The enzyme was purified

ity

agents

Both reagents

as an anionic

PST was purified

150-175

RESEARCH COMMUNICATIONS

modifying

on PST activity.

residue

Materials

AND BIOPHYSICAL

buffer,

out

in

pH 7.9;

BIOCHEMICAL

Vol. 78, No. 3, 1977

phenylglyoxal

(Sigma)

periments

out

at

assayed

for

residual

chemical (variable)

were removed, and then

the

for

at any given

time

at

various using

experiments

were included

ex-

preincubation

activity.

was calculated

was

times

the

and

radio-

p-nitrophenol

during

5 mM phosphate

residual

the

of enzyme and incubation

competition

against

in

preincubation

activity

appropriate

dialyzed

assayed

All

sulfotransferase

In the

After

prepared

Samples were withdrawn

or PAPS (variable)

step.

maining

by addition

25°C.

assay.

concentration)

enzyme preparation.

were initiated

carried

tion

(variable

and the

same buffer;

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

preincuba-

period

aliquots

buffer,

pH 7.40,

The percent

activity

relative

zero-time

to

re-

activity. Results

and Discussion

In preliminary rapidly tion

inactivate is

first the

rate

kinetics

resulted

phate ther

with

respect

for

posed earlier To provide

experiments modification

(2).

upon the

the

by Riordan

al -et --

further

evidence

reagent

specific

of

which

is

consistent

modification

was replaced

of

with

phos-

providing

borate

complex

furpro-

(2). that

was due to arginyl were performed

and

concentration

of PST was observed,

product-stabilizing

pseudo-

The inactivation

upon dialysis,

buffer

inactiva-

of 2,+butanedione

inactivation,

When borate

this

enzyme remaining

mechanism of 2,3-butanedione

no inactivation

evidence

2,3-butanedione

of

for

followed

concentrations

to be reversible

residues buffer

rates

was shown to

course

to active

was dependent

Increasing

the proposed

time

The inactivation

1.

in increasing

was observed

2,3-butanedione

A detailed

of inactivation

2,3-butanedione.

arginyl

PST.

shown in Figure

order

with

experiments

using

the

residue

PST inactivation modification,

phenylglyoxal,

for

1069

arginyl

another

residues

(7).

by similar chemical In Figure

Vol. 78, No. 3, 1977

BIOCHEMICAL

01

I 15

0

1 30

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0 45

I 60

PRElNCUBATlON

I 75

TIME

1 so

I 105

I 120

lmmutesl

Figure 1: Effect of varying concentrations of 2,3-butanedione on PST activity as a function of preincubation time. PST was incubated with 10 mM ( 0 ), 20 mM ( A ) or 40 mM ( 0) 2,3-butanedione. The PST activities were determined as described in the Materials and Methods Section.

2 is

shown a plot

maining

vs.

trations first

of the

preincubation

kinetics

with

The enzyme activity consistent

with

could the

modifying Evidence

recognition the

time

of phenylglyoxal. order

arginyl

log of the percentage

that site

respect

of varying

concen-

follows

pseudo-

enzyme remaining.

recovered

properties

re-

again

to active

be partially

after

of phenylglyoxal

dialysis, as an

(7).

an arginyl

residue

was obtained

enzymes two substrates

presence

The inactivation

general

reagent

in the

PST activity

from

protection

p-nitrophenol

1070

on PST is

acting

as an anion

experiments

using

and PAPS (Table

1).

Inclusi

Vol. 78, No. 3, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PREINCUBATION

TIME

(mmuresl

Figure 2: Effect of varying concentrations of phenylglyoxal on PST activity as a function of preincubation time. PST was incubated with 1 mM ( 0 ), 2 mM ( A ) or 5 mM (0 ) phenylglyoxal as described in the Materials and Methods Section.

of p-nitrophenol dione tion.

failed

in the preincubation to afford

However,

was included

have

experiments

shown that

certain

1071

enzyme from sulfate

complete

inactivadonor,

protection

of

was observed.

2,3-butanedione

enzymes contain

PST and 2,3-butane

charged

mixture,

by 2,3-butanedione using

of the

negatively

preincubation

PST from inactivation Recent

any protection

when PAPS, the

in the

step with

arginyl

and phenylglyoxal residues

as anion

Vol. 78, No. 3, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table

1

Substrate Protection of PST from Inactivation by 2,3-Butanedione

Additionsa

Residual Activityb after 90 min, 25O %

Concentration mM

None p-Nitrophenol

-4 40

p-Nitrophenol PAPS

37 34 30

0.48

PAPS

98 95

4.84

aThe standard preincubation mixture consisted of 2,xbutanedione (40 mM); borate buffer, pH 7.40 (50 mM); and the enzyme preparation in a total volume of 0.4 ml. The preincubation was carried out for 90 min at 25' after which the samples were dialyzed against phospH 7.40 (5 mM) for 4-5 hours. The phate buffer, samples were then assayed for residual activity using the methods described in the text. b PST activity remaining was calculated using as a reference a preincubation mixture treated under similar conditions except the 2,x-butanedione was not included.

recognition thought tidase

sites.

For

to be crucial A (12);

example,

in the

NADH binding

template

binding

to

viruses;

and substrate

the reverse binding

(l),

fructose-6-phosphate

kinase

(l),

and pyruvate

reported

donor

have arginyl

residues

arginyl cognition

here

is,

residues sites

could

to

creatine

(1).

(l),

list

the first

1072

hexo-

from the

utilize

PAPS

of enzymes which sites.

The data

evidence

in sulfate-transfer charged

RNA

C RNA

(14),

which

recognition

the negatively

(13);

3-phosphoglycerate

sulfotransferases

our knowledge,

carboxypep-

would appear

the

are

from type kinase

It

be added to

to

dehydrogenases

kinase

as anionic

participate for

to

sites

of peptides

transcriptases

kinase

here that

as a sulfate

reported

binding

recognition

to alcohol

kinase

results

these

that

enzymes as resulfate

donor

PAPS.

Vol. 78, No. 3, 1977

BIOCHEMICAL

AND BIOPHYSICAL

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Acknowledgements: The authors gratefully acknolwedge support for this project by grants from the National Institutes of Health (GM-22357) and the Kansas Heart Association. This work was done during the tenure of an Established Investigatorship of the American Heart Association. References 1.

2. 3.

4. 5.

6. 7. 8. 9. 10. 11. 12.

13. 14.

Riordan, J. F., McElvany, K. D. and Borders, 6. L. Jr., Science, 195, 884 (1977) Meek, J. L. and Neff, N. H.. J. Neurochz., 3, 1 (1973). Jenner, W. N. and Rose, F. A.; Bicohem. 3 135, 109 (1973). Eccleston, D. and Ritchie, I. M., J-- . Neuroc-., 21, 635 (1973). Braestrup, C., Nielsen, M. and Scheel-Kriiger, J., -J. Neurochem., 23, 569 (1974). Riordan, J. F., -Fed. -Proc. -Fed. -Amer. -* Sot -2& Exp w., 462 (1970). Takahashi, K., J. Biol. m., 243, 6171 (1968). Davis, D. C., Brochem. Pharmacol., 24, 975 (1975). Foldes, A. and Meek, J. L., Biochim. Biophys. m, 327, 365 (1973). Roy, A. B., Biochem. J., 74, 49 (1960). Balasubramanian, A. S., Spolter, L., Rice, L. I., Sharon, J. B. and Marx, W., Anal. Biochem., 21, 22 (1967). Riordan, J. F., Biochem., 12, 3915 (1973). Lange, L. G. III, Riordan, J. F. and Vallee, B. L., Biochem., 3, 4361 (1973). Borders, C. L., Riordan, J. F. and Auld, P. S., Biochem. Biophys. Res. Commun., 66, 490 (1975).

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