Evidence for involvement of arginyl residue at the catalytic site of penicillin acylase from Escherichiacoli

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vo1.173, No. 1,1990

Pages 317-322

November 30,1990

EVIDENCE

FOR INVOLVEMENT OF ARGINYL RESIDUE AT THE CATALYTIC OF PENICILLIN ACYLASE FROM ESCHERICHIA COLI 1 ASMITHA

A.

AND H E P H Z I B A H

PRABHUNE

SITE

SIVARAMAN

Division of Biochemical Sciences National Chemical Laboratory Pune - 411008, India

Received October 15, 1990

SUMMARY: Incubation of penicillin acylase from Escherichia coli with phenylglyoxal or 2,3-butanedione results in enzyme inactivation. Both benzylpenicillin and phenylacetate protect the enzyme against the inactivation, indicating the presence of arginine at or near the catalytic site. The reactions follow pseudofirst order kinetics and the inactivation kinetics indicate the presence of a single essential arginine moiety. ©1990AcademicPress, Inc.

Penicillin hydrolysis

acylase

of

penicillanic in

the

such

acid

have as

only

a

and

the

lin

acylase

among ~) In

few

The

have

been

reported

from

an

coli,

the (3),

see

has

of

is

the

been

(~)

and

enzyme

from

Kluyvera

key

intermediate

E.

In

be

coli

Benzylpenicilstudied

a heterodimer (~)

subunit

resembles (4)

the

mechanism

extensively to

69,000

citrophila

of

contrast,

reaction

most

M r

aspects

cloning

enzymes.

shown

an

Extensive

i).

the

the

6-amino-

industrial

and

Ref.

on

group

which

enzymes,

M r 20,500

formation

serine

the

of

the

an

kinetics

reagent

structurally

1 NCL

E. of

this

on

enzyme

studies

of

and

those

and

(2). from

Arthrobacter

(5).

from

activates

the

review

centers

catalyses

acid the

consequently of

coli

penicillins.

(for

structure

The lated

out

E.

being

semisynthetic

carried

rettgeri

viscosus

latter

gene

class

subunit

the

from

phenylacetic

acylase

containing

Proteus

to

immobilization

active

this

of

been

the

penicillin

3.5.1.11)

(6-APA),

manufacture

studies

(EC

benzylpenicillin

acyl-enzyme of

enzyme

the

enzyme;

a mole

No.

side of

chain the

has

catalyzed

phenylmethanesulfonyl

resembles

Communication

the

intermediate

been

postu-

reaction

(6).

fluoride, of

reagent

which

benzylpenicillin, completely

also in-

inactiva-

5012.

317

0006-2917(/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 173, No. 1, 1990 ting

a

mole

(3).

of

the

However,

inactivates about

the

rettseri

an

the

presence

of

of (8)

an

from

E.

the

enzyme serine

active (9)

or

from

P.

rettgeri

diisopropylfluorophosphate

coli

coli

(7)

residue

site has

casting

of been

ambiguity (7).

enzymes

The from

suggested

P.

from

studies.

this

communication

essential

arginine

MATERIALS

coli

essential at

E.

E.

reagent

the

tryptophan and

from

serine

weakly

inactivation In

enzyme

only

involvement

of

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AND

we p r e s e n t residue

in

evidence the

E.

coli

for

the

presence

enzyme.

METHODS

Phenylglyoxal and 2,3-butanedione were obtained from Fluka, benzylpenicillin and 6-aminopenicillanic acid (6-APA) were gifts from Hindustan Antibiotics Ltd., phenylacetic acid was o b t a i n e d from Aldrich, and DEAE-Sepharose CL-6B was f r o m Pharmacia. Escherichia coli NCIM 2 3 5 0 w a s o b t a i n e d from the National Collection of Industrial Microorganisms, Pune, and was m a i n tained routinely on n u t r i e n t agar slants.

Penicillin acylase. The enzyme was isolated from the extract of cells of E. coli grown at 26°C for 24 h under shake flask conditions in a medium of the following composition: yeast extract, 2 g; bactopeptone, 2 g; tryptone, 1 g; beef extract, 2 g; corn steep liquor (0.5 g/ml), 25 ml; K2HPO 4, 3 g; KH2PO., 0 . 3 g; NaC1, 3 . 5 g; (NHa)TSO a, 1 g; M g S O a . 7 H ~ O , - 0 . 2 g; pheny~acetic acid, 1 g; and wat6r-to-make to 1 L-after adjustm e n t o f pH t o 7 . 2 . Cells were harvested by c e n t r i f u g a t i o n , washed with 0.01 M potassium phosphate buffer, pH 7 . 5 and stored at -20°C. Cells (60 g wet weight) were thawed, suspended in 0.05 M potassium phosphate buffer, pH 7.5, (3 ml/g packed cells), cooled in an ice bath and disrupted by sonication (20 Kc,300 W) for a total period of i0 min. Cell debris was removed by centrifugation and all subsequent operations were carried out at 0-4°C. The clear cell-free extract was subjected to ammonium sulfate fractionationj and the fraction obtained between 0.3 0.8 saturation was c o l l e c t e d , dissolved in the minimum volume of 0.05 M potassium phosphate buffer, pH 7 . 5 , and dialyzed overnight against I00 volumes of buffer of the same composition. The dialysate was c l a r i f i e d by c e n t r i f u g a t i o n and applied on t o a DEAE-Sepharose C L - 6 B c o l u m n (3 x 75 cm) p r e v i o u s l y equilibrated with 0.0i M potassium phosphate buffer, pH 7 . 5 . The e n z y m e was e l u t e d using a linear gradient of potassium phosphate buffer, pH 7 . 5 (0.01 - 0 . I M). The eluant with enzyme activity was c o n c e n t r a t e d by u l t r a f i l t r a t i o n and loaded on a Sephadex G-200 column equilibrated with 0.05 M potassium phosphate buffer, pH 7 . 5 ; the fractions with penicillin acylase activity were pooled and concentrated by u l t r a f i l t r a t i o n .

318

Vol. 173, No. 1, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Enzym e assay. Penicillin acylase activity was assayed essentially as described by Balasingham et al. (10) by measuring the amount of 6-APA produced at 40°C using 4% b e n z y l p e n i cillin as substrate in 0.I M potassium phosphate buffer, pH 7 . 8 and 6-APA produced was measured with p-dimethylaminobenzaldehyde according to the procedure of Bomstein and Evans (II). One u n i t (U) o f e n z y m e a c t i v i t y is defined as the amount of enzyme catalyzing the hydrolysis o f i Dmol o f s u b s t r a t e in I min under the assay conditions. Lowry

Protein et al.

assay. Protein (12) using bovine

was determined serum albumin

Gel ~lectrophoresis. acrylamide gel electrophoresis using Tris-glycine buffer, (t3).

Sodium was pH 8.8

by the method as standard.

dodecylsulfate carried out as described

in

of

(SDS)-poly7.5% gels by Laemmlli

Treatment with arginine modifyi~ reagents. Penicillin acylase was incubated at 25°C with either phenylglyoxal (2.5-20 mM)in 0.05 M potassium phosphate buffer, pH 8.0! or 2,3-butaneaione (I0 - 40 mM) in 0.05 M sodium borate buffer, pH 8.0. The reaction system was shielded from light when 2,3-butanedione was used as the arginine modifying reagent (14). Enzyme incubated with buffers in absence of the modifying reagents served as controls. Aliquots were withdrawn at different time intervals for assay of enzymatic activity. Protection of the enzyme against inactivation arginine reagents was tested with benzylpenicillin-K phenylacetate. The compounds were tested at 50 mM centration and pH 8.0 and were added immediately addition of phenylglyoxal (20 mM final concentration) butanedione (40 mM~ final concentration.

by salt final before or

the and conthe 2,3-

RESULTS Enzyme vity

purification:

of

basis

The

27 U/mg p r o t e i n . of

purified The

SDS-polyacrylamide

polypeptide

bands

enzyme

had

preparation gel

of Mr~--,20,000

a specific

acti-

was homogenous

electrophoresis,

and Mr,-,70,000

on

only

being

the two

observed

on

staining. Inactivation cillin with

was

phenylglyoxal

potent wed

by

acylase

or

inactivator pseudo

being

residual than presence

residual

initial of

40

1). kinetics

in enzyme

Reagents: on

rates in

versus

upto

the

presence

test of

activity,

raM 2,3-butanedione

former

of

both

cases, of

period 20

mM the

coli

peni-

at

pH 8 . 0

being

a more

inactivation

time

being

319

E.

incubation

the

The

activity

linear

activity 5%

Modifying inactivated

2,3-butanedione,

(Fig.

first-order

logarithm reagents

Enzyme

rapidly

of

plots

of

the

contact

with

the

60

when

the

min

phenylglyoxal corresponding 26%.

follo-

was value

less in

Vol. 173, No. 1, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

IC

~ J.8

.e~

1.8

J,4

~ i.o T

1.6

i

i

2-0

2.4

I

0

-log (M) [ A. Phenylglyoxol

I-0

I

I-4

1"8

-leg (M)

2.0I B. 2~g- Butenedione

2.0

g

,~1.0 2

0 0

I

i

l

20

40

60

I01

0

I

I

i

20

40

60

Time(min)

Fig.

1.

Inactivation carbonyi The

of

penicillin

acylase

from

E.

coli

by

~-

reagents.

enzyme

potassium

(60

#8/ml)

phosphate

centrations

of

buffer

8.0)

(pH

was

incubated

buffer

(pH

phenylglyoxal and

or

varying

at

8.0] in

25°C

and

in

50

varying

50 mM

sodium

concentrations

mM

conborate

of

2,3-

butanedione (A)

Phenylglyoxai

(-i-), (B)

2,3-butanedione (-i-),

At

0

the

drawn Insets: with

30 mM

indicated for

mM

(-O-),

10 mM ( - ~ - )

of

mM

(-O-),

5 mM

10 raM ( - O - ) ,

20

mM

a n d 40 mM ( - X - ) .

of

time

aliquots

were

with-

enzyme activity.

Determination to

2.5

20 mM ( - X - ) .

0 raM ( - O - ) , (-D-),

periods

assay

respect

and

of

the

order

phenylslyoxal

(A)

of

and

the

reaction

2,3-butanedione

(B).

The was

first

reaction

determined

order

order from

rate

(n)

the

constant,

plot

with

to

respect of

Ka p p 320

the

the

logarithm

versus

the

arginine of

the

logarithm

reagent apparent

of

the

Vol. 173, No. 1, 1990 T a b l e 1.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Protection

of

inactivation

E,

coli

penicillin

by a r g i n i n e

acylase

specific

against

reagents

Treatment a

Enzyme a c t i v i t y b (% i n i t i a l activity)

None

100

Phenylglyoxal

(20 raM)

Benzyipenicillin

4

(50 mM) + p h e n y l g l y o x a l

Phenylacetate

(50 mM) + p h e n y l g l y o x a l

2,3-Butanedione

(20 raM)

95

(20 mM)

60

(40 mM)

Benzylpenicillin Phenylacetate

26

(50 mM) + 2 , 3 - b u t a n e d i o n e (50 mM) + 2 , 3 - b u t a n e d i o n e

(40 mM) 98

(40 ~M)

92

a T e s t compounds when u s e d w e r e a d d e d i m m e d i a t e l y b e f o r e t h e ~-carbonyl reagent. Enzyme (60 ~ g / m l ) was t r e a t e d at 25°C as i n d i c a t e d i n T a b l e and s a m p l e s w e r e w i t h d r a w n p e r i o d i c a i l y f o r a s s a y o f enzyme a c t i v i t y . bvalues

reagent and

obtained

after

concentration

of

n = 0.97

activity

results

penicillin

and

for

from

summarizes

the

acylase of

data

on by

n = 0.95

indicate

reaction

of

for

that

one

95%

phenylglyoxal loss

of

arginine

at the

end

against and

respectively values

inactivation

for of

inactivation

by

or

enzyme

per

mole

20

the

period absence

the

enzyme

by by

Table

i

inactivation

of

the

2,3-butanedione

in

the

the

enzyme

mM p h e n y l g l y o x a l

test

protection

reagents:

phenylacetate.

respectively

the in

of

raM) p r o t e c t e d

both

98%

of

arginine

protection

by and

by

phenylglyoxal

(50

inactivation

26~,

ponding

the

benzylpenicillin

butanedione,

and

of

inactivation

Benzylpenicillin

retained

value

acylase.

penicillin

against

the

2,3-butanedione

from

Protection

presence

60 m i n .

almost

and

fully

40

raM 2 , 3 -

of

initial

activity

being

of

60

compared

to

of

min,

substrate.

The

corres-

60%

against

and

92%

against

shown

in

phenylacetate phenylglyoxal

4%

are

2,3-butanedione.

DISCUSSION Penicillin sent

study

to

acylase be

from

inactivated

E.

cell

by 321

has

arginine

been

specific

the

reagents

presuch

Vol. 173, No. 1, 1990 as

phenylglyoxal

vation

indicate

enzyme

is

which

indicating, of

not

presence

that

would of

of

an in

that

charged

1

is

hitherto

essential

at

in

or

this

that

act

anionic

cofactors

near

greup

on an a r g i n y l

the

of

enzymes

has

The

residue

active

been

substrates

(15). E.

the

arginlne

residue

from

the

2,3-butanedione

on a n i o n i c

acylase

mole

of

protects

essential

arginine

enzymes

substrate

(10) and

an

per

phenylacetate,

inhibitor

of

inacti-

presence

product,

residue

of

reagent

The

the

involvement

depend

mole

phenylglyoxal

benzylpenicillin

enzymes

negatively

by

reported

require

add

of

competitive

The

extensively

those

or

arginine

been

of

kinetics

inactivation.

inactivation the

The

reaction

the

a weak

enzyme.

has

reported

list

as

that the

The

study

the for

benzylpenicillin

against

residue

2,3-butanedione.

that

functions

enzyme

site

and

required

substrate,

or

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

present

coli to

to

act

this

upon

a

molecule.

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Shewale, J.G., 24, 146-154.

2.

Bock, A., Wirth, R., and Buckel, P. ( 1 9 8 3 )

3.

Daumy, teriol.

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Barbero, J.L., Buesa, J.M., Perez-Aranda, A., and Garcia,

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Ohashi, H., Katsuta, Y., Hashizume, H., Hattori, H., Kamei, T., and Environ. Microbiol. 5.4, 2 6 0 3 - 2 6 0 7 .

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Konecny,

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Robak, 275.

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Mahajan, P.B., Bull. 2_5_5, 6 - i 0 .

G.O., 163,

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Process

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E.

K., Warburton, Biochim. Biophys.

Acta

(1983)

Biochem.

Hindustan

!!

Bomstein, 578.

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Lowry, O.H., Rosebrough, N.J., Farr, A.L., R.J. (i951) J. Biol. Chem. 1 9 3 , 2 6 5 - 2 7 5 .

13

Laemmlli,

14.

Levy, Chem.

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