Vol. 166, No. 2, 1990 January 30, 1990

BIOCHEMICAL

EVIDENCE

AND BIOPHYSKAL

RESEARCH COMMUNICATIONS Pages 904-908

FOR INVOLVEMENT OF 2 HISTIDINE RESIDUES THE REACTION OF AMPICILLIN ACYLASE Deog Jung Kim

and Si Myung

IN

Byun’

Department of Biological Science and Engineering Korea Advanced Institute of Science and Technology P.O. Box 150, Chongyang, Seoul 130-650, Korea Received

December

20,

1989

SUMMARY: The chemical modification of purified ampicillin acylase by Nbromosuccinimide and diethylpyrocarbonate resulted in time-dependent inactivation of the enzyme. Both substrates, ampicillin and 6-aminopenicillanic acid, protected the enzyme against inactivation, suggesting that the modification occurred near or at the active site. Amino acid analyses and other data indicated that two histidyl residues per subunit molecule were essential for catalytic activity. 01990 Academic Press, Inc.

Since Sakaguchi (EC

351.11)

demonstrated

in

preferred

chrysugenum,

penicillin

(Amp)

of two identical

and cephalexin, subunits.

this enzyme requires Determination

(2, 3). Penicillin

acylase has

been

G, penicillin according

to

site of the enzyme.

with a molecular

in particular,

is known

to be

a free amino

group

of the essential amino However, preparation

on the a-carbon

to an understanding

that donor.

in substrate binding

of the nature of the active

has been reported

in the catalytic mechanism

of ampicillin

should

of the acyl group

acid residues involved

no information

so far. The present report correspondence

weight of 146 000 and is composed

The substrate specificity was shown in our laboratory

amino acid residues that are involved since no purified

melanogenum,

both of which contain a side chain of D-phenylglycine

or in catalytic activity is fundamental

whom

activity

acylase, because it shows activity only for the synthesis of

(4). The enzyme is a glycoprotein

*To

enzyme

acylase are three types of acylases that are classified acylase from Pseudomonas

an interesting

available

the presence of penicillin

similar

substrates.

Ampicillin ampicillin

(1) first reported

Penicilliur72

to occur in many kinds of microorganisms

V and ampicillin their

and Murao

acylase from P.

concerning of ampicillin

melonogenum

describes the results of chemical

specific acylase, has been

modification

of

be addressed.

Abbreviations: NBS, N-bromosuccinimide; DEPC, diethylpyrocarbonate; 6-APA, 6aminopenicillanic acid; PGM . HCl, phenylglycinemethylester hydrochloride; Amp, ampicillin; PITC, phenylisothiocyanate; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis. 0006-291X/90 Copyright All rights

$1.50

0 1990 by Academic Press, Inc. of reproduction in any form reserved.

!I04

Vol.

166,

No.

2, 1990

purified

BIOCHEMICAL

ampicillin

AND

BIOPHYSICAL

acylase by N-bromosuccinimide

RESEARCH

(NBS)

and

COMMUNICATIONS

diethylpyrocarbonate

(DEPC) . MATERIALS

AND

METHODS

Ampicillin acylase was purified to homogeneity in our laboratory from Pseudomonns melnnogenrdm. Briefly. the purification procedure involved the successive fraction of the crude cell extract over columns of S-Sepharose, hydroxyapatite, CM-Cellulose C52, and CM-Sepharose. The purified enzyme had a specific activity of 956 units/mg protein, and its purity was confirmed by the techniques of 12% SDS-PAGE, analytical isoelectricfocusing and Protein PAK300 SW HPLC chromatography. 6-Aminopenicillanic acid (6-APA), Amp, and inhibitor compounds were obtained from Sigma. Phenylisothiocyanate (PITC), 6N hydrochloric acid and Amino Acid Standard H were purchased from Pierce. The hydrolytic activity of ampicillin acylase was determined by measuring the amount of 6-APA formed in a reaction mixture containing 5 mM Amp and enzyme in 10 mM sodium phosphate buffer (pH 6.0) (5). The Amp synthesized in the reaction mixture containing enzyme, 20 mM 6-APA and 60 mM phenylglycinemethylester hydrochloride (PGM* HCI) was determined calorimetrically at 320 nm according to the method described by Smith er al. (6). Chemical modification of the enzyme was carried out at 37’C in a reaction mixture containing enzyme, 1-7 FM NBS and 0.1-l mM DEPC in 10 mM sodium phosphate (pH 6.0). For studies on the protection of the enzyme against chemical modification, the enzyme was preincubated with different concentrations of 6-APA, PGM. HCI, and Amp, and modification was done as indicated in the table legend. Samples containing about 1.5 pg ampicillin acylase modified by NBS were dried under vacuum using a PICO-TAG Workstation and hydrolyzed with 6N HCl irk vocuo at 105’C for 24 h. The amino acid analysis was performed by reverse-phase derivatization of the protein hydrolyzates chroamtography with PITC precolumn (7-8).

RESULTS The chemical inhibitors activity. DEPC

modification

showed When

AND

of purified

Both

the

the enzyme

hydrolytic

and

of tryptophan,

DEPC

reacts with

that DEPC slightly

was incubated synthetic

tyrosine,

activities

significant

various of this

enzyme

cysteine and histidine

modifies

and amino histidine

groups

residues

at the active site. and Amp,

and Amp the

of NBS and

as shown were

in Fig. 1.

simultaneously

modification

It is well

known

that

However,

in proteins

with

it has also been shown

considerable

specificity

at a

effects of the three kinds of enzyme

whether

the modified

Because the DEPC

residues were located

inhibitor

reacted with

6-APA,

whereas NBS did not react with these substrates, the protective

exhibited

substrate PGM. HCI that

acid

acid side chains, such as imidazole,

(12-14).

The protective

(9-11).

effect of the substrates was tested against NBS inactivation. APA

amino

decrease in enzyme

concentrations

was observed

proteins having several amino

sulfhydryl,

acidic pH of 6.0 (15).

PGM. HCl

with

inactivation

substrates were next assayed to examine near or

acylase with various a

by NBS and DEPC. NBS has been shown to modify or cleave the functional

groups

phenolate,

ampicillin

that only NBS and DEPC caused

at pH 6.0, a time-dependent

inactivated

DISCUSSION

protective

displayed occurred

effects against NBS inactivation,

no such effect. This near

Table

or

at the

905

result provides active

site.

1 shows that 6but definite

Moreover,

the other evidence this

data

Vol.

166, No. 2, 1990

BIOCHEMICAL

AND BIOPHYSICAL

B

A

5

0

-

15

10

RESEARCH COMMUNICATIONS

1 5

0

1 15

10

TIYE(MlN)

TIME

(MIN)

Fig. Time course of inactivation of ampicillin acylase by NBS and DEPC. The enzyme was incubated at different concentrations of NBS and DEPC in 10 mM sodium phosphate buffer (pH 6.0) at 37% At intervals, aliquots were removed and assayed for enzyme activity on the hydrolysis of ampicillin as described in “Materials and Methods.” A. The NBS concentrations were: I, 0 pM; , 1 7 CM. B. The DEPC concentrations PM; o-,3 pM; O----o, 5 pM; m_) were: P---a , 0 mM; m , 0.1 mM; 04, 0.25 mM; q 4 , 0.5 mM; W, 1 mM.

(Table

1) elucidated

ampicillin

effects against NBS inactivation, at the binding

simultaneous

inactivation

site of 6-APA,

close enough The amino

residue

to influence

at the active

it is certain

that the modified

but not at that of PGM-

is located

HCl.

showed residue

site of

is

However, NBS

PGM . HCl,

of PGM * HCl.

acylase modified

with

NBS indicated

between the residual enzyme activity and the number

Table 1. Protective effects of substrates&the

site of

and synthetic activities with

near the binding

the hydrolysis

acid analysis of ampicillin

there is a correlation

residues

and Amp, but not PGM.HCl,

of the enzyme’s hydrolytic

suggests that the modified being

of the modified

acylase. Because the substrates 6-APA

protective located

the location

that

of modified

inactivation of ampicillin acylase by

Residual activity (S) Incubation time

(min)

NBS

NBS+ 6-APA 70 pM(350

5 10

21 14

15

14

70 43 43

(86) (80) (IJO)

$4)

NBS+ Amp 70 pY(350 57 43 43

(93) (86) (86)

UM)

NBS+ PCkHCl 70 LdA(350pY) 26

(26)

18 (16) 18

(18)

The enzyme was incubated at 37’C in the assay buffer with 7 pM NBS, in the presence of 6-APA (70 or 350 *M), Amp (70 or 350 +M), or PGM - HCl (70 or 350 PM). At intervals, aliquots were withdrawn and assayed for enzyme activity on the hydrolysis of Amp. Residual activity was corrected with the blank value in the absence of Amp substrate and expressed relative to the original enzyme activity.

Vol.

166,

No.

2, 1990

BIOCHEMICAL

0

1 HISTIDINES

AND

BIOPHYSICAL

2

RESEARCH

COMMUNICATIONS

4

3 MODIFIED

/

SUBUNIT

Fia. Correlation between the residual enzyme activity and the number of modified histidine residues. The enzyme (0.1 u,M) was incubated with 1, 3, 7 and 10 +M NBS in 10 mM sodium phosphate buffer (pH 6.0), at 37°C for 5 min. Each sample was hydrolyzed with 6N HCI in ~clcuo at 105’C for 24 h and analyzed as described in “Materials and Methods.” The unmodified enzyme was similarly analyzed without the addition of NBS.

histidine

residues per subunit.

per subunit molecule revealed from

As shown in Fig. 2, modification

of ampicillin

that two residues

acylase resulted in complete

out of four modified

histidine

the active site and that only two histidine

essential for the catalytic model

having

activity of ampicillin

an acyl enzyme intermediate

system using the a-amino showed

that ampicillin

hydrolase. proteins

group

capable of acting as proton

the histidine

residues,

be involved

in

intermediate.

Further

the catalytic mechanism

transfer

investigation

This result

of histidine

apart

molecule

citri.

reaction Our

activity as a-amino

are

a reaction

synthesizing

from Xunrhomonas

catalytic

residues

residues are located

residues per subunit

for the cephalexin

work

acid ester

is one of the functional

groups

donors and acceptors, there is a possibility

essential for the catalytic

proton

inactivation.

acylase. Kato (16) proposed

acid ester hydrolase

acylase had a similar

As the imidazole

of 4 histidine

in a reaction on the role

activity of ampicillin mechanism

having

the mechanism

that

acylase, may an acyl-enzyme

of the essential histidine

is necessary for understanding

of

residues

in

of the enzymatic

reaction. REFERENCES 1. 2. 3. 4. 5.

Sakaguchi, K. and Murao, S. (1950) J. Agric. Chem. Sot., Jpn. 23, 411-413. Vandamme, E. J. and Voets, J. P. (1974) Adv. Appl. Microbial. 17, 311-369. Hamilton-Miller, J. M. T. (1966) Bacterial. Rev. 30, 761-771. Shimizu, M., Masuike, T. and Fujita, H. (1975) Agr. Biol. Chem. 39, 1225-1232. Blasingham, K., Warburton, D., Dunnill, P. and Lilly, M. D. (1972) B&him. Biophys. Acta 276, 250-256. 907

Vol.

166, No. 2, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

6. Smith, J. W. G., Grey, G. E. and Patel, V. J. (1967) Analyst 92, 247-252. 7. Robert, L. H. and Stephen, C. M. (1984) Anal. B&hem. 136, 65-74. 8. Henning, S. (1985) J. Chromatogr. 350, 453-460. 9. Witkop, B. (1961) Adv. Protein Chem. 16, 221-285. 10. Vallee,, B. L. and Riordan, J. F. (1969) Annu. Rev. Biochem. 38, 733-794. 11. Lundblad, R. L. and Noyes, C. M. (1984) in Chemical Reagents for Protein Modification, Vol. II, pp. 47-72, CRC Press, Boca Raton, FL. 12. Miller, E. W. (1977) Methods Enzymol. 47, 431-442. 13. Muhlrad, A., Heigyi, G., and Toth, G. (1967) Acta B&hem. Biophys. Acad. Sci. Hung. 2, 19-29. 14. Burstein, Y., Walsh, K. A. and Neurath, H. (1974) Biochemistry 13, 205210. 15. Melchoir, W. B. and Fahrney, D. (1970) Biochemsitry 9, 251-258. 16. Kato, K. (1980) Agric. Biol. Chem. 44, 1083-1088.

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