Content uploaded by Robert A Bonomo
Author content
All content in this area was uploaded by Robert A Bonomo on Jul 04, 2020
Content may be subject to copyright.
Biochem. J. (1998) 333, 395–400 (Printed in Great Britain) 395
Kinetic analysis of an inhibitor-resistant variant of the OHIO-1 β-lactamase,
an SHV-family class A enzyme
Shan LIN*, Mary THOMAS*, David M. SHLAES*, Susan D. RUDIN*, James R. KNOX†, Vernon ANDERSON‡
and Robert A. BONOMO*§1
*Research Service, Department of Veterans Affairs Medical Center, 10701 East Boulevard, Cleveland, OH 44105, U.S.A., †Department of Molecular and Cell Biology,
University of Connecticut, Storrs, CT 06269-3125, U.S.A., ‡Department of Biochemistry, Case Western Reserve University, School of Medicine, 2109 Adelbert Road,
Cleveland, OH 44105, U.S.A., and §Geriatric CARE Center, University Hospitals of Cleveland, 12200 Fairhill Road, Cleveland, OH 44120, U.S.A.
The Met'* !Ile mutant of the OHIO-1 β-lactamase, an SHV-
family enzyme, is resistant to inactivation by β-lactamase in-
hibitors. Analysis of purified Met'* !Ile enzyme reveals that its
isoelectric point (pI 7.0) and CD spectrum are identical with
those of the OHIO-1 enzyme. Levels of β-lactamase expression in
Escherichia coli as determined by immunoblotting are similar for
OHIO-1 and Met'* !Ile β-lactamase. The kinetic constants of
the Met'* !Ile enzyme compared with OHIO-1 are smaller for
benzylpenicillin (Km¯6µM compared with 17 µM; kcat ¯
234 s−"compared with 345 s−"respectively) and carbenicillin (Km
¯3µM compared with 17 µM; kcat ¯131 s−"compared with
320 s−"respectively). For the cephalosporins cephaloridine and
7-(thienyl-2-acetamido)-3-[2-(4-N,N-dimethylaminophenylazo)-
pyridinium-methyl]-3-cephem-4-carboxylic acid (PADAC), a
similar pattern is also seen (Km¯38 µM compared with 96 µM
INTRODUCTION
The most common and important mechanism of bacterial
resistance to β-lactam antibiotics is the production of β-lactamase
enzymes. β-Lactamases are bacterial periplasmic enzymes that
hydrolyse β-lactam antibiotics into inactive acids. Four classes of
β-lactamases have been described. Classes A, C and D are serine
hydrolytic enzymes ; class B β-lactamases are zinc metalloenzymes
[1,2]. So far more than 190 distinct enzymes have been recorded
[3]. The diversity of these enzymes is matched only by their great
catalytic efficiency. The rapid spread and evolution of these
plasmid and chromosomally encoded enzymes have seriously
threatened our antimicrobial arsenal [4].
To combat the resistance conferred by β-lactamase enzymes
two strategies are employed. The first is to design or discover β-
lactam antibiotics that are able to escape hydrolysis yet still are
effective inactivators of penicillin-binding proteins (for example
the penicillinase-resistant penicillins and oxyiminocephalo-
sporins). The second is to couple a β-lactam antibiotic to a β-
lactamase inhibitor. These inhibitors irreversibly inactivate class
Aβ-lactamases. Ampicillin}sulbactam, amoxicillin}clavulanate,
ticarcillin}clavulanate and piperacillin}tazobactam are marketed
examples of this tactic.
Mechanistically, the β-lactamase inhibitor (I) first binds re-
versibly to the β-lactamase (Scheme 1). The enzyme is then
acylated (E–I). The acyl intermediate has three potential fates :
Abbreviations used: LB, Luria–Bertani ; PADAC, 7-(thienyl-2-acetamido)-3-[2-(4-N,N-dimethylaminophenylazo)pyridinium-methyl]-3-cephem-4-
carboxylic acid; pIEF, preparative isoelectric focusing.
1To whom correspondence should be addressed at Geriatric CARE Center (e-mail rab14!po.cwru.edu).
and 6 µM compared with 75 µM respectively; kcat ¯235 s−"
compared with 1023 s−"and 9 s−"compared with 50 s−"resp-
ectively). Consistent with minimum inhibitory concentrations
that show resistance to β-lactam β-lactamase inhibitors, the
apparent Kivalues, turnover numbers and partition ratios
(kcat}kinact ) for the mechanism-based inactivators clavulanate,
sulbactam and tazobactam are increased. The inactivation rate
constants (kinact ) are decreased. The difference in activation
energy, a measurement of altered affinity for the wild-type and
mutant enzymes leading to acylation of the active site, reveals
small energy differences of less than 8.4 kJ}mol. In total, these
results suggest that the Met !Ile substitution at position 69 in
the OHIO-1 β-lactamase alters the active site, primarily affecting
the interactions with β-lactamase inhibitors.
(1) hydrolysis of the active site ester resulting in the regeneration
of active enzyme and hydrolysed inhibitor; (2) the formation of
an irreversibly inactivated enzyme by secondary covalent modifi-
cation; and (3) undergoing a reversible change that generates a
transiently inhibited enzyme. This transiently inhibited enzyme is
called a tautomer, and the process of generating a transiently
inhibited enzyme is called tautomerization.
Point mutations in the TEM and SHV family of the class A β-
Scheme 1 Mechanism of activation of class A β-lactamase by inhibitors
Abbreviations: E, β-lactamase ; I, inhibitor ; E[I, Michaelis complex; E–I, acyl enzyme ; E–T,
tautomer ; P, product ; E–I*, irreversibly inactivated enzyme ; kft, forward tautomerization; krt,
reverse tautomerization.
396 S. Lin and others
lactamase are being described in the clinic and the laboratory
that alter the ability of various inhibitors to inactivate the
enzyme. Among the most common amino acid changes in TEM
that confer resistance to inactivation by mechanism-based in-
hibitors are mutations at positions Met'*, Arg#%% and Asn#(' [5].
So far the only inhibitor-resistant β-lactamases that have been
reported in the SHV family are the SHV-10 β-lactamase and
laboratory isolates of the SHV-1, SHV-5 and OHIO-1 variety
[6–10]. These include mutations at position 69 (Met'* !Ile) and
position 276 (Asn#(' !Gly) in the OHIO-1 β-lactamase, and
positions 130 and 244 (Ser"$! !Gly, Arg#%% !Ser and Arg#%% !
Cys) in the SHV family. Although the TEM family of enzymes
that possess resistance to β-lactamase inhibitors are the most
prevalent, it is feared that with the extensive use of oral and
intravenous β-lactam}β-lactamase inhibitor combinations there
will also be an increase in the prevalence of resistant SHV
enzymes found in the clinic. In this paper we describe in detail the
kinetic interactions of a class A β-lactamase of the SHV family
(the Met'* !Ile mutant of the OHIO-1 β-lactamase) that is
resistant to inactivation by the mechanism-based inhibitors
clavulanic acid, sulbactam and tazobactam.
MATERIALS AND METHODS
Bacterial strains and plasmids
Escherichia coli strain DH5α(supE44,∆lacU169)(Ø80
lacZ∆M15) hsdR17, recA1,endA1,gyrA96,thi-1,relA1) was
employed in the experiments described. The OHIO-1 and Met'*
!Ile β-lactamase genes were directionally subcloned into the
phagemid vector pBC SK (®) (Stratagene, La Jolla, CA, U.S.A.),
which encodes for chloramphenicol acetyltransferase (chlor-
amphenicol resistance), and transformed into E.coli DH5α[7–9].
Plasmids were isolated with a Wizard Miniprep Kit (Promega
Co., Madison, WI, U.S.A.). All bacteria were grown in Luria–
Bertani (LB) broth and agar containing the appropriate antibiotic
selection (20 µg}ml chloramphenicol or 100 µg}ml ampicillin
and 20 µg}ml chloramphenicol). Experiments in our laboratory
have shown that there is no effect of chloramphenicol on the level
of expression of β-lactam resistance. Restriction enzymes were
obtained from Promega (Madison, WI, U.S.A.) and United
States Biochemical (Cleveland, OH, U.S.A.).
Chemicals
Benzylpenicillin, carbenicillin, ampicillin and cephaloridine were
purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.).
Nitrocefin was purchased from Becton Dickinson Microbiolo-
gical Systems (Cockeysville, MD, U.S.A.). 7-(Thienyl-2-acet-
amido)-3-[2-(4-N,N-dimethylaminophenylazo)pyridinium-
methyl]-3-cephem-4-carboxylic acid (PADAC) is a Calbiochem
product (La Jolla, CA, U.S.A.). Clavulanate was kindly provided
by Smith Kline Beecham Laboratories (Bristol, TN, U.S.A.).
Tazobactam was provided by Lederle Laboratories (Pearl River,
NY, U.S.A.). Sulbactam was a gift from Pfizer (New York, NY,
U.S.A.). The chemical structures of these compounds are shown
in Figure 1.
Phenotypic characterization
Minimum inhibitory concentrations were determined on LB agar
plates with a Replicator (Chester, PA, U.S.A.) that delivered 10%
colony-forming organisms per spot. After inoculation each plate
was incubated for 18 h at 37 °C.
β-Lactamase purification
The OHIO-1 and Met'* !Ile β-lactamases were purified to
homogeneity by preparative isoelectric focusing (pIEF). E.coli
DH5αcontaining the OHIO-1 and Met'* !Ile β-lactamase genes
was grown in LB broth containing 100 µg}ml ampicillin and
20 µg}ml chloramphenicol. β-Lactamase was liberated by strin-
gent periplasmic fractionation with lysozyme by the method of
Johnson and Hecht [11]. After liberation of the β-lactamase,
crude lysates were clarified by passage through a Centricon filter.
Samples were next concentrated with an Amicon concentrator
with a 10 kDa cut-off.
pIEF was performed with a Multiphor II apparatus (Phar-
macia, Piscataway, NJ, U.S.A.) with a pH gradient of 6–8.
Ultradex gels were prepared with a concentrated protein sample
in accordance with specifications outlined by the manufacturer.
The pIEF gels were run overnight. β-Lactamase activity was
identified by using finely cut strips of filter paper applied to the
top of the gel matrix. The area where the β-lactamase was
focused was identified by soaking strips of paper with nitrocefin
and observing the colour change. Areas demonstrating β-lacta-
mase activity were removed from the gel; the active enzyme was
eluted from PEGG columns (Pharmacia, Piscataway, NJ, U.S.A.)
with 20 µM potassium phosphate buffer, pH 7.4. Ampholines
were removed by extensive dialysis in potassium phosphate
buffer. Samples were again concentrated with Amicon filters
(10 kDa cut-off) and purity was assessed by SDS}PAGE [15%
(w}v) gel]. Purified enzymes were stored at 4 °C until use. Specific
activity was determined as µmol of nitrocefin hydrolysed per min
divided by the total protein concentration.
To determine the precise pI of the purified enzymes, analytical
isoelectric focusing was performed using pH gradients of 3–11
and 6–8 using the Bio-Rad apparatus (Bio-Rad, Hercules, CA,
U.S.A.). β-Lactamase activity was detected by nitrocefin staining,
by the method of Vecoli et al. [12]; the homogeneity of the
sample was further assessed by staining with Coomassie Blue.
The first 20 residues of the purified wild-type enzyme were
sequenced at the Molecular Biology Core Facility at Case
Western Reserve University School of Medicine (Cleveland, OH,
U.S.A.).
Immunoblotting
Steady-state expression levels of OHIO-1 and Met'* !Ile β-
lactamases were determined by immunoblotting, with a modifi-
cation of the method developed by Petrosino and Palzkill [13]. E.
coli and DH5αcells containing the wild-type and Met'* !Ile β-
lactamases were grown in LB broth containing ampicillin and
chloramphenicol. Cell pellets were suspended in Tris}HCl buffer,
pH 7.4, and treated with lysozyme and EDTA. Crude lysates
were tested for their ability to hydrolyse nitrocefin. Equal
amounts of crude lysate (25 µg of protein) were resolved on
SDS}PAGE gels containing 15 %(w}v) polyacrylamide. Proteins
were next transferred to nitrocellulose membranes by electro-
blotting. The blot was probed by an anti-TEM polyclonal
antibody kindly provided by T. Palzkill. We have repeatedly
shown in our laboratory that this antibody cross-reacts with
SHV-1 type enzymes. Bound antibodies were revealed by using
anti-rabbit antibodies labelled with horseradish peroxidase and
with the Amersham Western Blot Kit.
Kinetic analysis
Protein concentration was determined by absorbance measure-
ments at 280 nM (∆ε33 400 M−"[cm−") [14]. A uniform con-
centration of enzyme was used for determining hydrolytic activity
397Inhibitor-resistant variant of the OHIO-1 β-lactamase
Figure 1 Chemical structures of β-lactam antibiotics used in this study
(typically 1.2 nM β-lactamase). β-Lactamase activity was moni-
tored with a spectrophotometer at 25 °C in 20 mM phosphate
buffer, pH 7.4.
Hydrolysis of the β-lactam substrates was performed at 25 °C
in a HP 8453 UV–visible spectrophotometer in a cell with a 1 cm
path length. The spectrophotometric assays of β-lactamase
activity were performed in 20 mM phosphate buffer, pH 7.4. The
molar absorption coefficients used were as follows: benzyl-
penicillin, ∆ε#%! 529 M−"[cm−"; carbenicillin, ∆ε#%!
400 M−"[cm−"; nitrocefin, ∆ε%)# 17400 M−"[cm−"; cephaloridine,
∆ε#'( 1000 M−"[cm−"; PADAC, ∆ε%'' 9590 M−"[cm−"; sul-
bactam, ∆ε#$'1780 M−"[cm−".
Initial rates () were determined from the first 5–10%of the
reactions at various substrate concentrations. The data were
fitted to the Michaelis–Menten equation by a non-linear least-
squares algorithm with the program ENZFITTER (Sigma).
Each determination of Vmax and the Kmpossessed an S.E.M. of
approx. 10%.
For the determination of the dissociation equilibrium constant,
Ki, the enzymes (1 nM) and stock concentrations of clavulanate,
sulbactam and tazobactam were preincubated for 10 min at
room temperature before the addition of a uniform concentration
of indicator substrate (100 µM nitrocefin), which served as a
competitor. All inhibition experiments were performed in 20 µM
phosphate buffer, pH 7.4. Standard plots of 1}against [I]
(Dixon plots) were prepared and the apparent Kiwas calculated.
To measure the turnover number (tn) of the enzyme for the
inhibitors, each inhibitor and enzyme were incubated at various
concentrations for 10 min and the residual activity was measured.
The turnover value was deduced from the extrapolated value for
100%inactivation from the plot of the remaining activity against
the ratio of [I] to [E] [15].
The partition ratios (kcat}kinact) for the inhibitors with wild-
type and mutant enzymes were determined by the titration
method, as described by Imtiaz et al. [16]. Various ratios of
inhibitor and enzyme concentrations were mixed and incubated
overnight at 4 °C (at least 18 h). The enzyme activity remaining
was assayed by monitoring the rate of hydrolysis of 100 µM
nitrocefin. Control samples containing enzymes without the
inactivator were similarly tested. Residual activity was then
398 S. Lin and others
assayed and a plot was made of the activity remaining against the
inhibitor-to-enzyme ratio.
The maximal inactivation rate constant, kinact, was obtained as
the absolute value of the slope of lnagainst time (t). A large
excess of nitrocefin was added and the remaining activity () was
measured. For clavulanate and tazobactam, kcat could not
be measured directly. For sulbactam a kcat (rate of hydrolysis)
was measured directly (∆ε#$' 1780 M−"[cm−").
CD measurements
CD spectra were recorded on a Jasco Model J-600 spectro-
polarimeter. The protein concentration was 6.9 µM for both the
wild-type and mutant enzymes in 20 µM potassium phosphate,
pH 7.4. We used quartz cuvettes with a 1 cm path length.
RESULTS
Minimum inhibitory concentration
To evaluate the phenotypic effect of the Met'* !Ile mutation in
the OHIO-1 β-lactamase enzyme, the wild-type and Met'* !Ile
β-lactamase genes were directionally subcloned into the phage-
mid vector pBC SK(®). This construct was transformed into
a uniform genetic background, E.coli DH5α. The wild-type and
Table 1 Minimum inhibitory concentration for E. coli DH5αwith and
without phagemid vector pBC SK(®)
OHIO-1 and Met69 !Ile are isogenic strains bearing the OHIO-1 and Met69 !Ile β-
lactamases.
Minimum inhibitory concentration ( µg/ml)
Ampicillin Ampicillin/clavulanate Ampicillin/sulbactam
DH5αwithout pBC SK(®)2%2/0 %2/0
DH5αwith pBC SK(®)2%2/0 %2/0
OHIO-1 4096 8/4 128/64
Met69 !Ile 2048 32/16 256/128
Figure 2 Purified protein preparations of the OHIO-1 and Met69 !Ile β-
lactamases resolved by SDS/PAGE [15 % (w/v) gel]
Left panel : lanes 1 and 2, purified OHIO-1 β-lactamase from different pIEF gels ; lane 3,
molecular mass markers (identified in kDa at the right). Right panel : lanes 1–3, purified Met69
!Ile β-lactamase from different pIEF gels; lane 4, molecular mass markers (identified in kDa
at the right).
Figure 3 Immunoblots of crude lysates of OHIO-1 and Met69 !Ile β-
lactamases resolved by SDS/PAGE [15 % (w/v) gel]
Left lane, OHIO-1 β-lactamase; right lane, Met69 !Ile β-lactamase.
the Met'* !Ile-bearing strains were tested against ampicillin,
ampicillin}clavulanate and ampicillin}sulbactam. The results are
summarized in Table 1. The Met'* !Ile β-lactamase expressed in
E.coli DH5αwas more resistant than the wild-type enzyme to
β-lactam}β-lactamase inhibitor combinations [7,8].
Purification and characterization of the OHIO-1 and Met69 !Ile
β-lactamases
The OHIO-1 and Met'* !Ile β-lactamase were purified to
homogeneity (Figure 2). The specific activity of samples prepared
in this manner increased 140–150-fold. The typical yield of
enzyme by our purification method was 20%. N-terminal amino
acid sequencing revealed that the mature protein began at ABL
residue Ser#' [16a]. Isoelectric focusing experiments demonstrated
that the pI values for OHIO-1 and Met'* !Ile β-lactamases were
both 7.0. The far-UV CD spectra of the OHIO-1 and Met'* !
Ile mutant β-lactamases at pH 7.4 were nearly superimposable.
Immunoblotting
β-Lactamase expression levels in iowere examined by immuno-
blotting; crude lysates were screened. Identical amounts of
protein were resolved on a SDS}PAGE gel. Immunoblots with
anti-TEM polyclonal antibodies revealed that the expression
levels of OHIO-1 and Met'* !Ile β-lactamases were similar
(Figure 3).
Kinetic analysis
The Kmvalues for benzylpenicillin, carbenicillin, PADAC and
cephaloridine were lower with the Met'* !Ile β-lactamase than
with OHIO-1 β-lactamase (Table 2). Similarly, the kcat values for
the Met'* !Ile mutant were lower for all substrates. The catalytic
efficiencies, defined as kcat}Km, were larger for some substrates
(penicillin, carbenicillin and PADAC) but smaller for others
(cephaloridine and nitrocefin). Nitrocefin was the only β-lactam
substrate that demonstrated a slightly larger Km.
The kinetic impact of the Met'* !Ile substitution against
irreversible inhibitors was quite pronounced (Table 3). The
dissociation equilibrium constant, Ki, for each inhibitor with the
wild-type and mutant enzymes was increased significantly. These
399Inhibitor-resistant variant of the OHIO-1 β-lactamase
Table 2 Kinetic parameters of OHIO-1 and Met69 !Ile β-lactamases
For all results the S.E.M. was ³10 %.
Substrate Enzyme Km(µM) kcat (s−1)k
cat/Km(µM−1[s−1)∆G‡
app (kJ/mol)
Benzylpenicillin OHIO-1 17 345 20
Met69 !Ile 6 234 39 #®1.7
Carbenicillin OHIO-1 17 320 19
Met69 !Ile 3 131 44 #®2.1
PADAC OHIO-1 75 50 0.6
Met69 !Ile 6 9 1.5 #®2.1
Cefaloridine OHIO-1 96 1023 10 1.3
Met69 !Ile 38 235 6 $1.3
Nitrocefin OHIO-1 11 115 9
Met69 !Ile 22 13 0.6 $6.7
Table 3 Kinetic parameters for inhibition
Inhibitor Enzyme Ki(µM) kinact (s−1) Turnover number kcat/kinact
Clavulanate OHIO-1 0.4 0.01 80 8
Met69 !Ile 10 0.002 2000 193
Sulbactam OHIO-1 17 0.001 40000 422
Met69 !Ile 68 0.0004 200000 5174
Tazobactam OHIO-1 0.30 0.01 400 4
Met69 !Ile 18 0.0016 2000 586
experiments were done with a concentration of inhibitor 1000-
fold greater than that of the enzyme.
The turnover number, the number of molecules of inhibitor
required to inactivate the Met'* !Ile β-lactamase, was greater
for each of the inhibitors than for the wild-type enzyme (Table
3). The most striking proportional increase in turnover number
activity was for clavulanate (25-fold). The turnover numbers
for sulbactam for the wild-type OHIO-1 β-lactamase and the
Met'* !Ile β-lactamase were both markedly greater.
The partition ratio, kcat}kinact, for each inhibitor was also
larger in the mutant (Table 3). In comparison with the wild-type
enzyme, the Met'* !Ile enzyme possessed a partition ratio 25-
fold greater for clavulanate, 5-fold greater for sulbactam and
more than 100-fold greater for tazobactam. The measured kcat
for sulbactam was 2.0 s−"for the Met'* !Ile enzyme, and 0.4 s−"
for the OHIO-1 β-lactamase.
DISCUSSION
The minimum inhibitory concentration results show that the Met
!Ile substitution at position 69 of the OHIO-1 β-lactamase
primarily affects the β-lactam}β-lactamase inhibitor combin-
ation. In normally susceptible E.coli DH5α, the Met'* !Ile
β-lactamase-expressing mutants are resistant to all the
mechanism-based inhibitors (Table 1).
The Ile substitution did not seem to change the isoelectric
point or CD spectrum of the β-lactamase. The nearly super-
imposable CD spectra suggests that the Ile substitution at
position 69 did not induce a major change in the secondary and
tertiary structural elements of the Met'* !Ile enzyme. This
suggests that the observed kinetic differences are not due to
major changes in the structure of the mutant β-lactamase. As
determined by immunoblotting, resistance to inhibitors does not
seem to be a consequence of increased levels of expression or
changes in stability of the β-lactamase enzymes.
Our analysis of substrate hydrolysis reveals that there is a
major effect of the Ile substitution on the catalytic behaviour of
the enzyme. For each of the substrates tested except nitrocefin,
there was a decrease in Km. Similarly, the kcat values were
decreased for all the β-lactams tested. Although kcat}Km(catalytic
efficiency) was increased for some substrates (penicillin, carbeni-
cillin and PADAC) and decreased for others (cephaloridine and
nitrocefin), the overall trend was that the Ile-substituted enzyme
turned over substrate more slowly than does the wild-type
enzyme. This difference is not unique to our mutant enzyme.
Farzaneh et al. [17] also showed that for the TEM variant of
Met'* !Ile, the kcat values are uniformly decreased. Although
we were not able to test it directly, this consistent decrease in
both kcat and Kmsuggests that the deacylation step might be the
rate-determining step. This is in accordance with the electrospray
MS findings of Saves et al. [18] that show that deacylation is the
rate-determining step, kcat, for penicillin by the TEM-1 enzyme.
A significant difference in inactivation was seen when the β-
lactamase inhibitors were tested against the wild-type and mutant
OHIO-1 enzymes. For each inhibitor, the dissociation equi-
librium constant, Ki, was increased. This increase was determined
with a concentration of inhibitor in 1000-fold excess. For
clavulanate and tazobactam the increases were 25-fold and 60-
fold respectively. For sulbactam the dissociation equilibrium
constant for the wild-type enzyme was already increased relative
to that for clavulanate and tazobactam; the increase over wild-
type for sulbactam with the Met'* !Ile enzyme was 4-fold.
β-Lactamase inhibitors are mechanism-based inactivators.
Hence they are recognized as a substrate by the enzyme [16]. The
turnover numbers of each β-lactamase inhibitor were uniformly
increased. The inactivation rate constant, kinact, was decreased
for clavulanate, sulbactam and tazobactam.
The partition ratio, the ratio of the amount of inhibitor that is
hydrolysed to the amount that inactivates the enzyme, was also
increased in Met'* !Ile compared with OHIO-1. From kcat}kinact,
the Met'* !Ile mutant enzyme hydrolyses sulbactam "tazo-
bactam "clavulanate. The wild-type enzyme hydrolyses sul-
bactam "clavulanate "tazobactam. The directly measured kcat
for sulbactam with the mutant enzyme is 5-fold that for the wild-
type (2.0 s−"compared with 0.4 s−"). The decreased efficiency of
inactivation and increased efficiency of hydrolysis of β-lactam
inhibitors indicate that there is a shift in the inhibition pathway.
The kcat}kinact of 8 for the OHIO-1 enzyme is significantly lower
than the kcat}kinact of 160 for TEM-1 [19]. However, it is in
400 S. Lin and others
accordance with the observation [6,15] that the IC&! of clavu-
lanate for SHV enzymes is lower than for TEM enzymes.
A unique feature of SHV-family enzymes is their intrinsic
resistance to sulbactam. Under the conditions of our experiments
we found that the sulbactam turnover numbers were 40 000 and
200000 for wild-type and inhibitor-resistant enzymes respec-
tively. Others also have found that turnover numbers are
elevated for sulbactam [15]. These experiments suggest that the
significant differences in the interaction of sulbactam with TEM
and SHV enzymes might be related to differences in tertiary
structure and to each agent’s mechanism of inhibition.
The contribution of the Met'* !Ile substitution to the overall
catalytic efficiency can be calculated for each substrate from the
results in Table 2. This was done with the equation :
∆G‡¯®RTln (kcat}Km)Met'* !Ile}(kcat}Km)OHIO-"
which calculates the difference in binding energy, ∆G‡, between
the wild-type and mutant enzymes in going from the free enzyme
plus substrate to the transition state. For penicillin, carbenicillin
and PADAC, the Met'* !Ile mutation decreases the free energy
barrier by approx. 2.1 kJ}mol. For nitrocefin and cephaloridine
∆G‡increases by 1.3 and 6.7 kJ}mol. In magnitude these changes
are relatively small (Table 2) and are similar to ∆G‡values that
we calculated for the Met'* !Ile mutant and the Met'* !Leu
mutant of TEM-1 [17,20].
On the basis of the modelling and energy minimization of the
crystal structure of the TEM-1 β-lactamase, Farzaneh et al. [17]
proposed that the Met'* !Ile substitution in TEM results in a
0.3–0.4 A
I
shift in the αcarbons from residues 69 to 72. Similar
shifts also occur in the side chain amide of Asn"(! (0.6 A
I
), the
hydroxy group of Ser(!, the carboxylic group of Glu"'' and the
oxygen atom of the catalytic water molecule. These authors
contend that this shifting causes a steric interaction between the
side chains of Asn"(! and Ile'*. They also state that a small
displacement (0.3 A
I
) causes the catalytic water molecule to be
displaced, resulting in a decrease in kcat and Km, and resistance to
inhibitors, particularly when deacylation is the rate-determining
step. In their analysis of the Met'* !Ile mutant, Chiabi et al.
have suggested that both hydrophobicity and steric constraints
are responsible for the altered activity [21].
The crystal structure of the OHIO-1 or an SHV β-lactamase
has not yet been determined. To understand the implications of
our findings, we previously constructed a three-dimensional
model of the OHIO-1 β-lactamase [8,9].
Residue 69 is located in a structurally conserved area of the
enzyme. A Met or Ile residue at this position has no direct
interaction with β-lactam substrates as they enter the active site
(Figure 3). The substitution of Ile, with its branched side chain,
having the β-methyl group position behind the B3 and B4 β-
strands and somewhat within the oxyanion pocket, is predicted
to affect the interaction of inhibitors with this enzyme. The 2-fold
greater hydrophobicity and steric bulk of the side chain of Ile
might perturb the shape and character of the oxyanion pocket
that binds the carbonyl group of the β-lactam ring. In addition,
a change in the position of the guanidinium group of Arg#%% on
B4 is expected. The effects of these changes are a decrease in the
catalysis of other β-lactams and an increase in the catalytic rate
constant for the hydrolysis of clavulanate, sulbactam and tazo-
bactam. In other words, because clavulanate, sulbactam and
Received 14 November 1997/26 March 1998; accepted 8 April 1998
tazobactam do not have large substituents at the C-6 position, the
catalysis of the β-lactamase inhibitors is increased. These com-
pounds might be better able to enter a more constrained active
site. Hence there is an increase in kcat}kinact. In contrast, the size
and character of the large R2 group at the C-6 position of the
other β-lactams decrease their turnover. The superimposable CD
spectra and the small changes calculated in the apparent binding
energy to the transition state are consistent with this model.
On the basis of the investigations of Farzaneh et al. [17],
Delaire et al. [20], Chiabi et al. [21] and the present study, the
kinetic impact of a mutation at position 69 seems to be similar in
both the TEM and OHIO-1 β-lactamases. However, it is possible
that important structural differences between TEM and OHIO-
1β-lactamases do exist. The catalytic differences between muta-
tions at position 69 and those at position 244 suggest that the two
sites affect substrate hydrolysis and confer resistance to inhibition
by very different mechanisms.
We thank Philip N. Rather and Louis B. Rice for critical review of this manuscript,
T. Palzkill for providing the anti-TEM polyclonal antibody, and Corina Rosiuta and
Deirdre Shedlow for their expert assistance.
REFERENCES
1 Ambler, R. P. (1980) Philos. Trans. R. Soc. London B 289, 321–331
2 Joris, B., Ghuysen, J. M., Dine, G., Renard, A., Dideberg, O., Charlier, P., Frere,
J. M., Kelly, J. A., Moews, P. C. and Knox, J. R. (1988) Biochem. J. 250, 313–324
3 Bush, K., Jacoby, G. A. and Medeiros, A. A. (1995) Antimicrob. Agents Chemother.
39, 1211–1233
4 Medeiros, A. A. (1997) Clin. Infect. Dis. 24 (suppl. 1), S19–S45
5 Knox, J. R. (1995) Antimicrob. Agents Chemother. 39, 2593–2600
6 Prinarakis, E. E., Mirriagon, V., Tzelepi, E., Gazouli, M. and Tzouvelekis, L. S. (1997)
Antimicrob. Agents Chemother. 41, 838–840
7 Bonomo, R. A., Currie-McCumber, C. and Shlaes, D. M. (1992) FEMS Microbiol. Lett.
92, 79–82
8 Bonomo, R. A., Dawes, C. G., Knox, J. R. and Shlaes, D. M. (1995) Biochim. Biophys.
Acta 1247, 113–120
9 Bonomo, R. A., Dawes, C. G., Knox, J. R. and Shlaes, D. M. (1995) Biochim. Biophys.
Acta 1247, 121–125
10 Giakkoupi, P., Tzelepi, E., Legakis, N. J. and Tzouvelekis, L. S. (1998) FEMS
Microbiol. Lett. 160, 49–54
11 Johnson, B. H. and Hecht, M. H. (1994) Biotechnology 12, 1357–1361
12 Vecoli, C., Prevost, F. E., Ververis, J. J., Medeiros, A. A. and O’Leary, G. P. (1983)
Antimicrob. Agents Chemother. 24, 186–189
13 Petrosino, J. F. and Palzkill, T. (1996) J. Bacteriol. 178, 1821–1830
14 Edelhoch, H. (1967) Biochemistry 6, 1948–1954
15 Bush, K., Macalintal, C., Rasmussen, B. A., Lee, V. J. and Yang, Y. (1993)
Antimicrob. Agents Chemother. 37, 851–858
16 Imtiaz, U., Billings, E., Knox, J. R., Manavathu, E. V., Lerner, S. A. and Mobashery, S.
(1993) J. Am. Chem. Soc. 115, 4435–4442
16a Ambler, R. P., Coulson, A. F. W., Fre
're, J.-M., Ghuysen, J.-M., Joris, B., Forsman, M.,
Levesque, R. C., Tiraby, G. and Waley, S. G. (1990) Biochem. J. 276, 269–272
17 Farzaneh, A., Charlie, E., Peduzzi, J., Barthelemy, M., Labia, R., Blazquez, J. and
Baquero, F. (1996) Antimicrob. Agents Chemother. 40, 2434–2436
18 Saves, I., Burlet-Schlitz, O., Maveyraud, L., Samama, J.-P., Prome
!, J.-C. and Masson,
J.-M. (1995) Biochemistry 34, 11660–11667
19 Imtiaz, U., Billings, E., Knox, J. R. and Mobashery, S. (1994) Biochemistry 33,
5728–5738
20 Delaire, M., Labia, R., Sammama, J.-P. and Masson, J.-M. (1992) J. Biol. Chem.
267, 20600–20606
21 Chiabi, E. B., Peduzzi, J., Farzaneh, S., Barthelemy, M., Sirot, D. and Labia, R. (1998)
Biochim. Biophys. Acta 1382, 38–46
