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.
REFERENCES 1.
Shewale, J.G., 24, 146-154.
2.
Bock, A., Wirth, R., and Buckel, P. ( 1 9 8 3 )
3.
Daumy, teriol.
4.
Barbero, J.L., Buesa, J.M., Perez-Aranda, A., and Garcia,
5.
Ohashi, H., Katsuta, Y., Hashizume, H., Hattori, H., Kamei, T., and Environ. Microbiol. 5.4, 2 6 0 3 - 2 6 0 7 .
6.
Konecny,
7.
Kutzbach, C., and Rauenbusch, Physiol. Chem. 3 5 4 , 4 5 - 5 3 .
8
Robak, 275.
9
Mahajan, P.B., Bull. 2_5_5, 6 - i 0 .
G.O., 163,
SivaRaman,
{1981)
and
and
(1989)
Mc'Call,
Process
(1985)
E.
K., Warburton, Biochim. Biophys.
Acta
(1983)
Biochem.
Hindustan
!!
Bomstein, 578.
12
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.
15.
Riordan, Science
U.K.
(1970)
J.F., McElvany, 195, 884-885.
(1965)
227,
and K.D.,
322
Analyt.
Z.
Pol
28,
Antibiot.
D., Dunnill, P., and A c t a . 27__~6, 2 5 0 - 2 5 6 .
W.G.
Nature
H.M., Leber, P.D., 238, 3654-3659.
Bac-
Hoppe-Seyler's
Balasingham, M.D. ( 1 9 7 2 )
and Evans,
J.
307-112.
(1_974)
(1981)
P.S.
~,
10
J.,
G., Lan8, G., 20, 135-139.
T., Abe, S.N., Kajiura, Yano, M. (i988) Appl.
Letts.
A.
Borkar,
A.S.
Biochem.
Buitrago, G.G., Mendez, E., J.L. ( 1 9 8 6 ) G e n e 4~9, 6 9 - 8 0 .
Biotechncl.
Szewczuk, and
H.
Schmid, G., Shumacher, FEMS M i c r o b i o l . Letts.
Danley, D., 1279-1281.
J.
M.,
and
Lilly,
Chem.
3_~7, 5 7 6 -
and
Randall,
680-685.
Ryan, and
E.M.
(1963)
Borders,
J.
C.L.
Biol. (1977)
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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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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Szewczuk, and
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Danley, D., 1279-1281.
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