(Received for publication, September 23, 1994)
From the
Using a random, combinatorial scheme of mutagenesis directed
against the conserved SDN region of TEM -lactamase, and selective
screening in ampicillin-plates, we obtained the N132D mutant enzyme.
The kinetic characterization of this mutant indicated relatively small
effects compared to the wild-type. Both pK
and
pK
for catalysis were decreased about 1 unit
relative to the pK`s for the wild type. This effect was predominantly
due to changes in K
. In contrast to the
wild-type, the pH-rate profiles of the mutant showed that K
for several side chain-containing
penicillin substrates increases when the pH is above 5.5.
6-Aminopenicillanic acid, which lacks a side chain, did not show this
effect. With benzylpenicillin, ampicillin, and carbenicillin, k
for the mutant showed a similar pH dependence
as the wild type. With 6-aminopenicillanic acid, k
for the mutant was greater than that for the wild type. The
nature of the 104 side chain may affect the environment of
Asp
; double mutants N132D/E104X (where X can be Q or N) are unable to confer antibiotic resistance to
bacterial cells. The computed contact interactions from modeling
substrate complexes between benzylpenicillin or 6-aminopenicillanic
acid with the N132D mutant confirmed the importance of the protonation
state of residue Asp
for the complex stability with side
chain-containing substrates. The data indicate that the contact between
the side chain of residue 132 and the substrate is relevant for the
ground state recognition, but because of close contact with several
important groups in its neighborhood, residue 132 is also indirectly
involved in the catalytic step of the wild-type enzyme.
Class A -lactamases are serine hydrolase members of a group
of enzymes that destroy
-lactam antibiotics by hydrolyzing the
sensitive four-membered
-lactam ring. TEM
-lactamase from Escherichia coli is the most common plasmid-mediated enzyme of
this class. Crystal structures of the
-lactamases of Staphylococcus aureus (Herzberg, 1991), Bacillus
licheniformis (Knox and Moews, 1991), and, recently, E. coli TEM
-lactamase (Strynadka et al., 1992; Jelsch et al., 1993) are known at high resolution. In addition, there
is crystallographic work with some mutant variants (Herzberg et
al., 1991; Knox et al., 1993), includ-ing one trapped in
the acyl-enzyme intermediate (Strynadka et al., 1992). Also,
there are reports about the structure of trapped intermediates of a
class A enzyme in complex with an inhibitor molecule (Chen and
Herzberg, 1992) or with a transition state analog (Chen et
al., 1993).
The generally accepted minimum kinetic scheme for
-lacta-mase is shown in ,
where EA represents the acyl-enzyme. For such a scheme,
and
The role of specific amino acid residues in the enzyme-catalyzed
process is not fully understood. It has been shown that besides serine
70, the transiently acylated residue, both lysine 73 and glutamic acid
166 are important for the catalytic mechanism. Site-directed
mutagenesis performed at these positions and in others resulted in
enzyme variants with drastic loss of activity (Ellerby et al.,
1990; Delaire et al., 1991; Brannigan et al., 1991;
Lenfant et al., 1991). Recent kinetic (Adachi et al.,
1991; Escobar et al., 1991) and structural (Strynadka et
al., 1992; Knox et al., 1993) data indicate that the role
of Glu involves its acting as a general base catalyst on
water poised to attack the acyl enzyme carbonyl and thus facilitate
deacylation. In spite of several possible routes that have been
proposed (Herzberg and Moult, 1991 and references therein), the
mechanism of the acylation half of the catalytic reaction remains
unclear. Studies by Jacob and collaborators (Jacob et al.,
1990a, 1990b) on the conserved triad SDN in Streptomyces albus
-lactamase showed that Asn
in this enzyme
stabilizes the transition state rather than having a role in ground
state binding.
To understand the role of several conserved residues
of the active site of TEM-lactamase, we have used random
combinatorial mutagenesis and selective screening approaches (growing
bacterial cells on ampicillin plates), in order to obtain protein
variants that remain catalytically active. In this work, we present
data from one such variant, Asp for Asn in position 132, that may help
define the role of the contact between the Asn
residue
and the substrate side chain.
Figure 1:
Structure of -lactam
antibiotics.
Figure 2:
k pH
profiles for the N132D mutant (open squares) and the wild-type (closed circles)
-lactamase with various substrates. A, benzylpenicillin; B, ampicillin; C,
carbenicillin, and D, 6-aminopenicillanic acid. All reactions
monitored at 30 °C. All the data, except that of C, were
fit to a standard ionization curve using . Data of C was fit to .
Figure 3:
K pH
profiles for the N132D mutant and the wild-type enzyme. The panels are
the same as described in the legend to Fig. 2.
Figure 4:
k/K
pH profiles for the N132D mutant and the wild-type enzyme. The
panels are as in Fig. 2. All the data were fitted to a standard
ionization curve using . The data for the wild-type enzyme
with carbenicillin as a substrate (C) gave a bad fit to , since the shape of the profile was not appropriate for
this type of analysis.
The pK values of the acidic limb
(pK
) for k
had values
similar to those of the wild-type, and the pK
s of
the basic limb (pK
) were slightly displaced to
lower values ( Table 3and Fig. 2).
With 6-APA as
substrate, the k of the mutant had higher values than the wild type. With this substrate, both
pK
and pK
for the mutant
enzyme had values very similar to the wild type ( Table 3and Fig. 2).
With carbenicillin as a substrate, the wild-type k showed an anomalous pH dependence, similar to
that shown for the Bacillus cereus
-lactamase (Hardy et al., 1984). Our mutant enzyme displayed a slightly
different behavior which seems to fit better with the same modified
equation () used in the work on the B. cereus
-lactamase.
The behavior of K for the mutant can be
separated in two groups, depending of the substrate used: (i) with
benzylpenicillin, ampicillin, and carbenicillin as substrates, K
increases at pH around 5.5 when compared with
wild-type values, which remain low even at pH 7.5. Around pH 5.0, the K
of the mutant enzyme approaches wild-type values (Fig. 3, A-C). (ii) With 6APA as a substrate, the K
obtained for the mutant was higher than the
wild-type value (an increase of about 5-fold) and that value remains
stable for a broader pH range when compared with other penicillins (Fig. 3D).
When compared at the pH optima, the
catalytic efficiency (k/K
)
of the mutant was around 30-50% of the wild-type efficiency,
depending on the substrate ( Fig. 4and Table 2). The pH
optimum of the mutant was shifted down by about 1 pH unit relative to
the wild-type enzyme. This reflects a decrease of about 1 in
pK
and 0.5 in pK
for the
N132D mutant relative to the values for the wild-type (Table 3).
With carbenicillin as substrate, the pH dependence of k
/K
of the N132D mutant was
different from that of the wild-type enzyme. Data from the N132D mutant
with this substrate fit very well to the usual equation ().
When tested with nitrocefin, an activated cephalosporin substrate,
the mutant enzyme had a k/K
value around 2-3% of the wild-type enzyme when compared at
the pH optima (k
/K
WT
= 4.2
10
;mz
s
and k
/K
mutant
= 8.5
10
;
mz
s
).
Herzberg and Moult(1987) and Moews et al.(1990)
working with different class A -lactamases have proposed that the
amide group of the Asn
side chain acts as an H-bond donor
to the carbonyl group of the substrate side chain, and recently
Strynadka et al.(1992), working with TEM
-lactamase, have
shown this same contact. Also, the side chain of Asn
forms a strong hydrogen bond with that of Lys
and is
in contact with a water molecule involved in a proposed replenishment
mechanism of the hydrolytic water molecule (Knox and Moews, 1991) (Fig. 5). Additionally, in the B. licheniformis enzyme
Asn
is in close contact with the side chain of the
Asn
residue. In the TEM and S. aureus enzymes,
this close contact between the side chains of residues 132 and 104
might be replaced by a contact between the side chain of Asn
and the main chain carbonyl of Glu
or
Ala
, respectively (Fig. 5). As show in Fig. 5, the side chain of residue 104 turns away from residue
132 in the TEM enzyme but not in the B. licheniformis
-lactamase structure.
Figure 5:
Stereoview of some active site residues
(TEM coordinates kindly provided by Dr. M. N. G. James). Potential
hydrogen bonds are indicated by dashed lines. Some
crystallographic water molecules are included as solid
spheres. In position 104 (Glu in the E. coli TEM
-lactamase), we superimposed the structurally homologous
residues of S. aureus (Ala
) and B.
licheniformis 749/C (Asn
) enzymes (coordinates 3BLM
and 4BLM, respectively; Protein Data Bank, Brookhaven National
Laboratory). The deacylating water is labeled 321. Penicillin
G (BPE) is positioned for
-face acylation by reactive
Ser
as modeled by Moews et al. (1990).
Jacob et al. (1990a, 1990b)
working with S. albus -lactamase variants, where
Asn
has been replaced with Ala or Ser, have proposed a
role for this residue in transition state stabilization rather than in
ground state binding. In that work, k
was the
most affected parameter, and the authors suggested an effect mainly in
the acylation step. Those workers reported that the replacement N132A
results in a very poor enzyme against all substrates tested and that
the alteration N132S results in a slightly impaired enzyme against
penicillin substrates. This latter mutant enzyme also showed poor
activity against substrates that are lacking a side chain (6-APA), and
finally, both mutant enzymes showed lower activities against
cephalosporins as compared with penicillins.
We believe that our
variant, with a less size-disruptive modification (Asn to Asp),
provides an interesting probe to determine the role of the contact
between the Asn residue and the substrate side chain,
both in ground state recognition and in transition state stabilization.
As can be seen in the Fig. 2, A-C, with all
the side chain-containing penicillin substrates tested, k for the mutant decreased about 2-fold when
compared with the corresponding wild-type values. At low pH (around pH
5.5), this behavior could be explained by the mutation decreasing the
rate of both acylation and deacylation steps to a similar extent. This
is sufficient to explain the decrease of k
and
the similarity, at this pH, of K
to wild-type
values (Fig. 3, A-C). At pH higher than 5.5, K
is sharply increased, whereas in the wild-type
enzyme K
values remain low even at pH 7.5 (Fig. 3, A-C). Thus, at pH 5.5 the effect of the
substitution of aspartate for asparagine leads to destabilization of
the transition state, whereas at higher pH the effect is on the ground
state. Since N132D k
for this kind of substrate
follows a pH response similar to that shown by the wild-type enzyme (Fig. 2, A-C), it implies that the perturbations
of k
/K
against pH in the
mutant relative to the wild type (Fig. 4, A-C) is
due mostly to the effect on the K
component of K
.
With a side chain-lacking substrate, such as
6-APA, both k and K
of the
mutant are higher than the respective parameters of the wild-type
enzyme. In kinetic terms (using ), this might be explained
by a better deacylation rate (k
), which would
result in an increase in k
and K
, as observed. The behavior of K
for the N132D mutant with 6-APA is different when compared with
that of the other substrates (Fig. 3D). K
for this substrate stays at a low value between
pH 5.5 and 7.5.
Our results also indicate that the side chain of
Glu may perturb the environment of the aspartate in
position 132. Analytical isoelectrofocusing results (data not shown)
show that the N132D mutant has essentially the same pI as the wild-type
enzyme. Thus, it appears that because of the proximity of glutamate 104
and aspartate 132 (Fig. 5), in the N132D mutant either or both
of these carboxylic side chains have a significantly altered
pK
. Substitutions in the residue position 104 of
the N132D mutant produce enzyme variants unable to confer ampicillin
resistance at the lower antibiotic concentrations tested as shown by
the MIC analysis (Table 1).
To rationalize the differences
between the kinetic properties of the mutant and wild-type enzymes, we
must analyze the possible consequences of contacts lacking in the
mutant. We assume that the contact between the Asp side
chain and the substrate is formed only at low pH. At this pH (<5.5)
there is no significant difference between the wild-type enzyme and the
N132D mutant, as shown in Table 2. We postulate that the contact
with Lys
is not seriously affected, and the effects
proposed for the acylation step (k
) might be
explained by subtle changes in the optimal geometry of that contact in
response to the mutation. The effects proposed in the deacylation step (k
) might be explained by assuming that the mutant
has lost the contact with the water molecule (Fig. 5), impairing
the replenishment mechanism of the hydrolytic water. Finally, the
suggested improvement in the deacylation step for side chain-lacking
substrates (6-APA) might be explained by the presence of water in the
extra space available in the active site with this kind of substrate,
permitting the replenishment of the hydrolytic water.
The modeling
results suggest that side chain-lacking substrates are more tolerant
than side chain-containing ones to the subtle conformational changes in
the active site as result of the extra negative charge introduced
(Asp modeled in ionized form). The results suggest better
contact distances between benzylpenicillin and the active site of the
mutant with Asp
modeled in its protonated state (Table 4). This is in agreement with the activity showed by the
mutant enzyme at low pH. However, we obtained a very unstable complex
when this same substrate was docked in the active site of the mutant
with Asp
modeled in its ionized form. With 6-APA, the
modeling results show that the calculated distances between the docked
substrate and the mutant protein, irrespective of the protonated state
of residue 132, are closer to those shown by the wild-type enzyme (Table 4). Again, this results are in close agreement with the
N132D activity pH response for this substrate.
Our data show both
similarities and differences with the work reported by Jacob et al. (1990a, 1990b). The main difference is the mutant activity against
6-APA. The present study shows that 6-APA was the least affected
substrate in terms of the efficiency of the mutant compared with the
wild-type enzyme with the same substrate. The k for the N132D mutant was higher than for the wild-type
enzyme, and in agreement with the results of the S. albus work, there was a distinct increase in K
. The
N132D TEM enzyme showed activity levels, against penicillin substrates,
comparable with those shown by the N132S S. albus mutant and
also had a marked preference to hydrolyze penicillins as compared with
cephalosporins. We note that the alterations made in the S. albus
-lactamase are more disruptive than the one present in our
mutant. The loss of the contact with Lys
(expected for
both Ala or Ser mutants) and also the loss of the contact with the
substrate (perhaps just in the Ala mutant, as suggested by the authors)
results in a very poor enzyme (N132A) or in a slightly impaired enzyme
(N132S). The proposed effect of both mutations only in the acylation
rate could be rationalized if the extra space available in the mutation
site could be filled with water molecules ready to carry out the
suggested replenishment of the catalytic water, leaving the deacylation
rate unaffected.
In summary, we propose that the contact between the
Asn residue side chain and the carbonyl group of the
substrate side chain is only relevant for ground state recognition.
However, the present results and those of Jacob et al. (1990a,
1990b) show that due to additional contacts of Asn
with
different groups of the active site, it is also indirectly involved in
the catalytic mechanism. Mutations in Asn
could affect
both the acylation rate by impairing the interaction of this residue
with Lys
, and the deacylation rate by altering the control
of the replenishment of the hydrolytic water as suggested by the
contact of Asn
with a water molecule of a train leading
from the solvent to the interior of the active site.