Vol. 185, No. 3, 1992 June 30, 1992

EVIDENCE

BIOCHEMICAL

AND BIOPHYSICAL

FOR THE INVOLVEMENT OF TRYPTOPHAN OF GLUTATHIONE Jun

Nishihiral*,

'Second

Department

istry,

School Faculty

of of

of

Nishi'

Ishibashi', and

Received

May 15,

060,

Sakai',

Department

3 Department

Sciences,

Sapporo

SITE

3

Kumazaki

2 First

and

Pharmaceutical

IN THE ACTIVE

Masaharu

Takashi

Biochemistry,

Medicine,

38

S-TRANSPERASE P

Teruo

Shinzo

RESEARCH COMMUNICATIONS Pages 1069-l 077

of

Hokkaido

of

Biochem-

Biochemistry, University,

Japan

1992

SUMMARY: Glutathione S-transferase P (GST-P) exists as a homodimerit form and has two tryptophan residues, Trp28 and Trp38, in each subunit. In order to elucidate the role of the two tryptophan residues in catalytic function, we examined intrinsic fluorescence of tryptophan residues and effect of chemical modificaThe quenching of intrinsic tion by N-bromosuccinimide (NBS). fluorescence was observed by the addition of S-hexylglutathione, a substrate analogue, and the enzymatic activity was totally lost To identify whensingle tryptophan residue was oxidized by NBS. whichtryptophanresidueisinvolved in the catalytic function, each tryptophan was changed to histidine by site-directed mutagenesis. Trp28His GST-P mutant enzyme showed a comparable enzymatic activTrp38His mutant neither was ity with that of the wild type one. bound to S-hexylglutathione-linked Sepharose nor exhibited any These findings indicate that Trp38 is important GST activity. for the catalytic function and substrate binding of GST-P. 0 1992 Academic Press,Inc.

Glutathione family of of *To

of

S-transferases

catalyzing

electrophilic Mr

the

conjugation

compounds

45,000-50,000

whom correspondence

(GSTs)

(1,2).

that

of

glutathione

They

consist

should

[EC 2.5.1.181

exist

of

two

represent

a

to

a variety

as dimeric

proteins

identical

or

non-

be addressed.

Abbreviations: GST, glutathione S-transferase; GSH, glutathione; CDNB, 1-chloro-2,4-dinitrobenzene: IPTG, isopropyl-p-D(-)-thiogaNBS, N-bromosuccinimide; PCR, polymerase chain lactopyranoside; reaction: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 0006-291X/92

1069

$4.00

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

identical

subunits

fication

of

terminal

amino

isozymes Amino the

three

acid

sequences

in

the

er,

it

is

still study, the

fluorescence tryptophan

substituted mutagenesis, residues

cDNAs

(5).

considered

clear

function

quenching

methods.

residues tryptophan which

was participated

revealed in

deduced

from

chemical

in with that

the

catalytic

(5,6).

tryptophan

to

identify

catalytic

one

howev-

resi-

modification

histidine only

and involv-

mechanism of

Furthermore,

EXPERIMENTAL

the

residues

chemical

using

the

divides

conjugation,

involvement

residues

and

arginine

important

detail

involved

been

Tryptophan,

glutathione

we demonstrated

catalytic

GSTs have

as

of

on the

amino

p, and SC. the

of

to

specificity,

classification

many of

mechanism

not

a,

classi-

according

substrate

This

of

of

been

catalytic

this in

have

proposed

homology,

classes

RESEARCH COMMUNICATIONS

a species-independent

been

(4).

sequences

acid

ing

dues

sequence

into

aspartic

has

reactivity

nucleotide

In

isozymes acid

AND BIOPHYSICAL

Recently,

(3).

the

immunological

any

BIOCHEMICAL

185, No. 3, 1992

and whether

function, by

of

we

site-directed

two

tryptophan

function.

PROCEDURES

Materials The following materials were obtained from commercial sources: S-hexylglutathione, S-hexylglutathione-linked Sepharose from Sigma (St. Louis, USA): reduced glutathione (GSH) from Yamanouchi 1-chloro-2,4-dinitrobenzene (CDNB) from Tokyo (Tokyo, Japan); IPTG, isopropyl-~-D(-)-thiogalactopyranoKasei (Tokyo, Japan); side and N-bromosuccinimide (NBS) from Wako pure chemicals BamHI from Takara Shuzo (Kyoto, Japan). All (Tokyo, Japan); other chemicals were of analytical arade. Expression and purification of glutkhione S-ttansferase P A fragment between two AccI sites of GST-P cDNA clone (located at 13 nucleotides upstream from ATG initiation codon and 3'-end of the cDNA clone, pGP5) was inserted into BamHI site of the expression vector pET3a using BamHI linkers (7,8). This plasmid (pETGST-WT) was used for transformation of the E. coli BL21(DE3)LysS. The bacteria produced the GST-P protein fused with 18 amino acids, encoded by vector and linker sequences, at the amino terminus (9). The glutathione S-transferase P (GST-P) was isolated to homogeneity from E. coli transformed by GST-P cDNA. In brief, E. coli transformed with pET3a plasmid containing GST-P cDNA were cultured and GST-P expression was induced by addition of IPTG (0.5 mM). After further 2 hr incubation, the E. coli were disrupted by a French pressure cell disrupter at 1000 psi. The homogenate was centrifuged at 105,000 x g for 1 hr, and the 1070

Vol.

185, No. 3, 1992

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

supernatant was used as the cytosolic fraction. GST-P was purified by Sephadex G-100 column chromatography and S-hexylglutaGST thione affinity column chromatography as described (10). activities were measured by the method of Habib et al. (11). ConcenSDS-PAGE was carried out by the method of Laemmli (12). tration of GSTMr 50,270) was spectrophotometrically determined by using I$$ = 10.5. Molar concentration was estimated by amino acid composition analysis. The composition after hydroly110 "C, 24 hr) was detersis (6 N HCl containing 0.05% phenol, mined on a Hitachi 835 amino acid analyzer.

Measurement

of

tryptophan

intrinsic

fluorescence

A solution of GST-P in potassium phosphate buffer (10 mM, pH 6.5) was excited at 290 nm, and intrinsic tryptophan fluorescence emission was measured at 340 nm in the presence and absence of Shexylglutathione, which bound to the active site (3), using a Hitachi 500F spectrofluorometer.

Chemical

modification

of

tryptophan

with

NBS

GST-P was treated with NBS (1 mM) in borate buffer (0.1 M, pH 8.0) at 30 "C for 30 min, during which the reaction was monitored in the spectral range from 240 to 320 nm using a Hitachi 220A The reaction was terminated by removing excess spectrophotometer. NBS by Sephadex G-25 column chromatography. Determination of the number of the oxidized tryptophan residues (n) was calculated by using the following equation from the decrease of absorbance at 280

nm

(13).

n = (SA x 1.31

x Mr x V)/(W

x E)

111

&A, difference absorbance at 280 nm resulting from NBS modification: Mr, molecular weight of the subunit of GST-P (25.1 kDa); V, reaction volume (ml); W, weight of the enzyme (mg); E, moiecuiar extinction coefficient of a tryptophan residue (5500 M cm); empirical factor, 1.31.

Construction

of

mutant

GST-P plasmid

by site-directed

mutagenesis

Site-directed mutagenesis was carried out by "Splicing by overlapped extension (SOE) method" using the polymerase chain reaction according to Vallete et al. (14). Firstly, two PCR products of pETGST-WT template, 5'- and 3'halves of GST-P cDNA overlapping each other by mutated region, were separately made. The primers used were ET5 (5' outside primer) and mutated inverse sequence for 5'-half amplification, and mutated forward sequence and ET3 (3' outside primer) for 3'-half amplification (Table I). The products were separated from excess primers by agarose gel electrophoresis. Secondly, two PCR products were mixed and used as the template for second PCR reaction with ET5 and ET3 primers. In this reaction, some of the molecules of the first PCR products were hybridized with mutated overlap region and this hybrids were filled up by polymerase and served as a template. The full Table

I.

Oligonucleotide primers directed mutagenesis

Primer ET5 ET3 Trp28His Trp38His Underline:

used for

Sequence ACATATGGCTAGCATGACTG GCTAGTTATTGCTCAGCGGT GGGCCAGAGCCACAAGCAGGAGG CCTCCTGCTTGTXCTCTGGCCC CATAGATGTCCACCTTCAAGGCT AGCCTTGAAGGTGGACATCTATG position

mutated. 1071

site Note 5' outside 3' outside forward inverse forward inverse

Vol.

BIOCHEMICAL

185, No. 3, 1992

AND RIOPHYSICAL

RESEARCH COMMUNICATIONS

length mutated products were digested with BamHI, inserted into pET3a vector and transformed BL21(DE3)LysS as described above. The site specific tryptophan changes were confirmed by dideoxyEach mutant expressed at the protein level was sequencing (15). confirmed by SDS-PAGE. The Trp28His mutant of GST-P was purified by the same procedure as described for the wild type.

RESULTS

Expression

and purity

Expression mg of

the

The final

of

of recombinant GST-P

GST-P was very

purified

enzyme

enzyme

preparation

SDS-PAGE

(Fig.

1).

tion

14.5

Wol/min/mg

was

comparable

with

01

that

were

efficient

in

obtained

from

showed

The specific

(+)

protein.

of

This rat

more liter

the

final

specific

340

than

20

culture.

homogeneity

placenta

300

02

one

an apparent

activity

of GST-P from

which

upon

prepara-

activity

was

(16).

380

Wave Length (nm)

Fig.

1.

SDS-PAGE of the of IPTG induced

wild type E. coli;

Fig.

2.

Effect of S-hexylglutathione on the intrinsic fluorescence of tryptophan residues. GST-P (0.5 mg) in 1 ml of potassium phosphate buffer (10 mM, pH 6.5) was excited at 290 nm. Fluorescent spectra from 290 to 430 nm were determined in the presence of S-hexylglutathione from 0 to 400 fl at the interval of 40 m. Inset: Fluorescence quenching at 340 nm was plotted against the concentration of S-hexylglutathione.

1072

GST-P. 2, final

1, cytosolic fraction enzyme preparation.

Vol.

185,

No.

Effect

3,

BIOCHEMICAL

1992

of S-hexylglutathione

The intrinsic the

wild

type

saturated

at

result

suggests

vicinity

of

GST-P

constant

of

S-hexylglutathione

constant

and

on the

that

only

the

2). of

30 % (Fig.

addition

of

S-hexylglutaInset).

the was

was comparable

of

The

may exist

Furthermore, GST-P

residues

The quench-

2,

residue

with

Kd value

by the

concentration

site.

fluorescence

tryptophan

(Fig.

one tryptophan

active

@4. This for

Effect

from

about

COMMUNICATIONS

tryptophan

analogue

depending

added

RESEARCH

quenched

a glutathione

thione

360

derived

GST-P was significantly

was increased

about

BIOPHYSICAL

on intrinsic

fluorescence

S-hexylglutathione, ing

AND

in

dissociation

estimated

with

the

the

to

be

inhibition

GST activity.

of chemical

modification

of tryptophan

residues

by NBS on

GST activity NBS selectively

oxidizes

alkaline

pH and

residues

are

Decrease

of

absorbance

decrease

in

enzymatic

to

GST-P

(Fig.

at

all

one

due in

the

this

one

matic single

tryptophan

in

catalytic

Site-directed Two Trp38His,

of

of

by

of

Fig.

that

not

residue,

equation

of

the

[l]

(13).

a concomitant manner

of

NBS

observed

subunit

the

shown).

one

was

almost

tryptophan

excessive (Fig.

modified

but

with

weak

tryptophan

was finally

only

S-hexylglutathione

(data

or

3A).

addition

residue

the

activity

was calculated

neutral

a dose-dependent

residue

(Inset

tryptophan

activity

the

tryptophan

even

presence

in

No enzymatic

it

was oxidized

using

run was observed

activity

oxidized

Furthermore,

280

at

The oxidized

analyzed at

3A).

residue

oxytryptophan.

quantitatively

when

completely

produces

tryptophan

amount

3B).

In

GST-P showed These

not

the

results other

resiof

addition,

the

full

enzy-

indicate one,

NBS

that

a

participates

function.

mutagenesis of l'rp28 and Trp38

species

of

by replacing

GST-P each

mutants, tryptophan 1073

designated with

as histidine

Trp28His by

and

site-di-

Vol.

185,

No.

3,

1882

BIOCHEMICAL

240

280

AND

320

240

Wave

Fig.

rected

3.

To determine

mutagenesis. in

GST-P

showed

mutant type

the

COMMUNICATIONS

320

(nm)

enzyme,

the

the

same

Trp38His

II.

done

because

Trp28

enzymatic

the

and Trp38 Trp28His

activity

neither

as

expressed

parameters Vmax

(mM)

GSH Type

of

Although

mutant

Kinetic Km

Trp28His Trp38His

residue

function.

almost

Enzyme

anot

280

which

catalytic

Table

Wild

Length

RESEARCH

Modification of tryptophan residues by N-bromosuccinimide. A, NBS was added to the wild type GST-P (0.36 mg) dissolved in 1 ml potassium phosphate buffer (10 mM, pH 6.5). Molar ratio of GST-P to NBS, a, 1 : 0; b, 1 : 4; Inset: Quantitative C, 1 : 8; d, 1 : 12; e, 1 : 16. residues relationship between oxidized tryptophan subunit) and GST activity on the wild (mol/mol GST-P type GST-P. B, NBS (GST-P : NBS = 1 : 20 in molar was added to the wild-type GST-P (0.36 mg) with ratio) (f) or without (g) S-hexylglutathione (10 mM).

participates

wild

BIOPHYSICAL

(runol/min/mg)

CDNB

0.15

+ 0.02

1.15

+ 0.01

14.5

f 0.11

OS"

faoeo4

1.25

f

11.4

2

the

enzyme

activity

1074

0.03

was not

detected.

0.16

the the

Vol.

BIOCHEMICAL

185, No. 3, 1992

enzymatic

activity

Sepharose

in

(Table is

nor

spite

II).

essential

was adsorbed

by

significant

induction

of

These for

results

the

AND BIOPHYSICAL

indicate

catalytic

RESEARCH COMMUNICATIONS

S-hexylglutathione-linked of

that

function

GST-P

Trp38,

of

but

proteins not

Trp28,

GST-P.

DISCUSSION Tryptophan tant

residue

role

in

hydrophobic

stabilizing

not

in

garding

dynamical and

active

with

has

demonstrated

that

site

and

examining

the

combination

indole

been of

the of

is

not

an impor-

virtue the

role

chemical

In

GST-P

in

the

residue

is

charge-

tryptophan

information

re-

The precise

acid

clear. 38 of

of

structure.

amino

of

or

derive

protein

residue

this

within

study,

was

located

it

was

in

the

the

catalytic

of

tryptophan

residue

modification

and

site-directed

fluorescence

the

by however

to

still

a crucial

intrinsic with

play

property

this

tryptophan

had

ring,

utilized

microenvironment GST-P

conformation

fluorescence

movement

of

to

protonation-deprotonation

The

protein

site

active

of

(17,18).

residue

considered

protein

related

system

role

generally

characteristic

usually relay

is

function

by in

mutagenesis. Human placental and as

has the

tophan

two

GST has

tryptophan

same case

with

residues

on

but less

it

GST-P the

the

other

ing

to

did

indicated

that

one

study

polypeptides residue

between

and

Trp38,

The structure GST was

applicable

not

specify

tryptophan

shielded

was in

solvent.

placental are

(19).

homology

the

Based

GST-P,

to

case

similar

the of

1075

results

GST-P.

subunit

the

primary of Since

et

residue,

solvent polar

a

Arduini

was embedded

more

tryp-

using

tryptophan

aqueous

and

the

GST and the

the

flexible on

each

by

GST-P

each

around

study

residue

from

in

with

investigated

phosphorescence report

the

Trp28

placental

Their

polar

% sequential

residues,

temperature-dependent a1.(20).

85.6

site

in

the

whereas expos-

structures

phosphorescence the

active

site

Vol.

185,

No.

needs

3, 1992

to

with

BIOCHEMICAL

be exposed

substrates,

ble

and

more

may

stabilize

bond

and

aqueous

the

native

iar

expected

the

Trp28

of

these

of

to from

but

chemical

not

with Trp28,

of

x GST from

with

respect In

the

class

to

catalytic

function

to

tion.

to For

it function

by this

method

Trp28

the

from

by NBS,

howev-

reagent

in

contrary,

sug-

in

the

aspect

of

microenvironments

depend

is

shielding

be located

site

exposing

on their

residue

stabilize

to

and

x GST (21), in

the

pecul-

residues

in

the

which

lung

study at

had

result

in

had on

mutagenerevealed

the

active

already

that site

and

analy-

demonstrated

contacted supports

the

based

Crystallographic

sulfonate, This

clarify

mechanism

this

of

residues

Site-directed

function.

role

to

cysteine

GST-P was located

porcine

the

used

catalytic

in

catalytic

examine

was effectively

histidine

(22).

higher

conclusion,

or

residue hydrogen

chemical

on

x GSTs may

glutathione

Trp28

reaction

site

the

GSH analogue,

with

the

result,

histidine

sis

not

modification with

flexi-

hand,

site

modifications.

for

but

this

through

other

was employed

class

was essential

that

class

be involved

tryptophan

of

in

this

site

hydrophobic

This

This of

believed

Trp38,

interaction

be located

On the

chemical

mutagenesis

function

of

efficient

active

reacted

two

non-essentiality

sis

the

The different

residue.

results

to

GST-P may

phase.

its

COMMUNICATIONS

differences.

tryptophan

the

in

RESEARCH

As a function,

the

3).

Trp28

Site-directed

been

in

residues

of

for

interaction.

(Fig.

aqueous

catalytic

in

located

form

BIOPHYSICAL

site.

As shown

structural

the

is

tryptophan

that

around

Trp38 active

be

phase.

the

solvent

polar

to

both

to

the

hydrophobic

er,

gests

to

a substrate

considered

AND

our

with

Trp38

present

data

conformation. was indicated of is

that

GST-P, not

though

the

chemical

further

investigation

reaction on the 1076

was essential

the

understood

three-dimensional

enhance

Trp38

detail

yet.

This

structure of role

of

glutathione of

Trp38,

to

chemical residue the

may

active conjugaprecise

Vol.

185, No. 3, 1992

structural and nuclear

analyses magnetic

BIOCHEMICAL

of

the

AND BIOPHYSICAL

active

resonance

are

site under

RESEARCH COMMUNICATIONS

by X ray

crystallography

way.

REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Chasseau, L. F. (1979) Adv. Cancer Res. 29, 175-274. Jakoby, W. B., and Habig, W. H. (1980) in Enzymatic Basis of Detoxication (Jakoby, W. B., ea.), Vol. 2, pp. 63-94, AcademPress New York. i:nnervik: B. (1985) Adv. Enzymol. 57, 357-417. Mannervik, B., Alin, P., Guthenberg, C., Jensson, H., Tahir, M. K., Warholm, M., and Jornvall, H. (1985) Proc. Natl. Acad. Sci. USA, 82, 7202-7206. Mannervik, B., and Danielson, U. H. (1988) CRC Crit. Rev. Biochem. 23, 283-337. Armstrong, R. N. (1987) CRC Crit. Rev. Biochem. 22, 39-88. Studier, F. W., and Moffatt, B. A. (1986) J. Mol. Biol. 189, 113-130. Rosenberg, A. H., Lade, B. N., Chui, D. S., Lin, S. W., Dunn, J. J., and Studier, F. W. (1987) Gene 56, 125-135. Sugioka, Y., Kano, T., Okuda, A., Sakai, M., Kitagawa, T., and Muramatsu, M. (1985) Nucleic Acids Res. 13, 6049-6957. Tu, C. P. D., Weiss, M. J., Li, N. Q., and Reddy, C. C. (1983) J. Biol. Chem. 258, 4659-4662. Habig, W. H., Pabst, M. J. and Jakoby, W. B. (1974) J. Biol. Chem. 249, 7130-7139. Laemmli, U. K. (1970) Nature 227, 680-685. Spande, T. F., and Witkop, B. (1967) Methods in Enzymol. 11, 498-532. Vallete, F. E., Mege, E., ReiSS, A., and Adesnik, M. (1989) Nucleic Acids Res. 17, 61-68. Messing, J. (1983) Methods in Enzymol. 101, 20-78. Tamai, K., Shen, H., Tsuchida, S., Hatayama, I., Satoh, K., Yasui, A., Oikawa, A., and Sato, K. (1991) Biochem. Biophys. Res. Commun. 179, 790-797. Keil-Dlouha, V., and Keil, B. J. Mol. Biol. (1973) 81, 381394. Lowe, G., and Whitworth, A. S. Biochem. J. (1974) 141, 503515. Kano, T., Sakai, M., and Muramatsu, M. Cancer Res. (1987) 47, 5626-5630. Arduini, A., Strambini, G., Di Ilio, C., Aceto, A., Storto, and Federici, G. (1989) Biochim. Biophys. Acta 999, 203&%. Kong, K., Inoue, H., and Takahashi, K. (1991) Biochem. Biophys. Res. Commun. 181, 748-755. Reinemer, P., Dirr, H. W., Ladenstein, R., Schaffer, J., Gally, O., and Huber, R. (1991) EMBO J. 10, 1997-2005.

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