(Received for publication, September 23, 1994)
From the
Using a random combinatorial mutagenesis of TEM -lactamase,
directed against residues potentially involved in substrate
discrimination, followed by selection on third generation
cephalosporins, we obtained the double mutant E104M/G238S.
Additionally, by using cloning strategies and site-directed mutagenesis
we constructed the individual single mutants and also the single
modification E104K and the double mutant E104K/G238S, which broaden the
specificity of clinically isolated TEM
-lactamase variants. The
kinetic characterization of the purified double mutant E104M/G238S and
its single counterparts E104M and G238S was carried out. The single
mutant E104M exhibited increased k
values
against all substrates tested. K
values
remained similar to the values shown by the wild-type enzyme. The
mutation at E104M was responsible for the increased hydrolysis rate
against cefuroxime shown by the double mutant E104M/G238S. The effect
of mutation G238S varied more pronouncedly, depending on the substrate.
In general, a lower K
was observed, but
also a decreased k
. The double mutant
E104M/G238S exhibited a higher hydrolytic rate against cefotaxime
compared with the corresponding single mutations. We observed nearly a
1000-fold greater k
/K
for the double mutant than for the wild type. This
improvement in catalysis was the consequence of increased k
and decreased K
values. Computed contact interactions from modeling
substrate complexes show reliable results only for benzylpenicillin.
The modeling results with this substrate confirmed the observed enzyme
activities for the different single and double mutants. Analysis of the
apparent coupling energies, as calculated from the kinetic parameters
of the single and double mutants, showed that the quantitative effect
of a second mutation on a single mutant was either absent, additive,
partially additive, or synergistic with respect to the first mutation,
depending on the substrate analyzed.
Class A -lactamases constitute a rich source of information
about the relationship between the amino acid sequence and the function
of the protein (Ambler et al., 1991). The class A, C, and D
-lactamases represent three large families of active site serine
enzymes (Coutere et al., 1992). Several crystallographic
structures of class A enzymes are known (Herzberg, 1991; Knox and
Moews, 1991; Jelsch et al., 1993). In addition, there is
crystallographic work with some mutant variants (Herzberg et
al., 1991; Knox et al., 1993), including 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).
Because of the extensive use of -lactam
antibiotics, variant enzymes have emerged that are capable of
hydrolyzing an extended spectrum of substrates, including third
generation cephalosporins (Jacoby and Medeiros, 1991). The
understanding of this extended spectrum mechanism is crucial for
gaining insight in the design of new and better drugs. However, because
only a few previous studies have included biochemical descriptions of
the extended spectrum mutant enzymes (Sowek et al., 1991;
Huletsky et al., 1993; Lee et al., 1991), more work
is needed in order to dissect the individual contributions of the
substituted residues present in the variants to the recognition of
different substrates.
In this work we present data related to
determining the contributions of individual residues to the substrate
specificity of TEM -lactamase. We describe a new specificity
mutant altered in positions 104 and 238, obtained through random
combinatorial mutagenesis and selective approaches, and then we dissect
the individual contributions of each residue in order to analyze the
role of different parts of the active site in the substrate
discrimination process.
Figure 1:
Structure of
-lactam antibiotics.
The mutant E104M/G238S, and its single amino acid replacement counterparts E104M and G238S, were purified to greater than 90% homogeneity as determined by SDS-PAGE and isoelectric focusing-PAGE. Mutant enzymes with the G238S alteration were less stable than the others. Because of questions of stability, most studies were performed within a few days of the final purification step.
Hydrolysis rate studies for
each of these antibiotics with the different enzymes resulted in the k values shown in Table 2. The single
variant E104M produces an increase in k
over
wild-type values against all the substrates tested, whereas the G238S
mutant shows an increase in k
only for
cefotaxime.
It is clear from Table 3that the G238S
substitution increased the affinity of the enzyme for a variety of
-lactam substrates, with the exception of cefuroxime and to a
lesser extent to cefotaxime. The substitution E104M, on the other hand,
had little effect on the affinity for substrates. It is interesting to
note that with cefuroxime none of the mutations affected K
, and with cefotaxime the major effect on K
occurred only in the double mutant.
When
catalytic efficiencies of the -lactamases were compared, it became
evident that all the mutants were better enzymes against penicillin
substrates as compared with cephalosporin substrates (Table 4).
However, for the double mutant E104M/G238S, cefotaxime and
cephaloridine became better substrates than 6-aminopenicillanic acid.
As shown in Table 4, the variant E104M is a better enzyme than
the wild-type against all the substrates tested. The modification G238S
was detrimental for the catalytic efficiency against all substrates
tested except cefotaxime. It is important to note that the double
mutant catalytic efficiency against cefotaxime is nearly 1000-fold
greater than of the wild type.
Our results demonstrate the lack of
predictability of a particular mutation on the expected modification of k or K
for a particular
substrate. For instance, the single mutant G238S shows decreased k
and improved (decreased) K
against all substrates tested, excepting cefotaxime (for which k
is increased and K
remains similar to the wild-type value) and cefuroxime (K
remains similar to the wild-type value),
whereas the E104M modification increases the k
value against all substrates tested but had little effect on K
. However, this same alteration, present in the
G238S context, caused a significant decrease in K
for some substrates but little effect on others!
In a manner similar to Carter et al.(1984), we decided to analyze our results using the mutant cycle displayed in Fig. 2a. If replacement of different side chains induces no structural changes in the enzyme or enzyme-substrate complex their effects will be independent, and the overall change in interaction energy of the enzyme-substrate transition state in double mutants will be the sum of the corresponding terms for the two single mutants.
Figure 2:
a, the changes in interaction energy of
enzyme and transition state are represented by the G terms. For example,
G
is the difference
in Gibbs-free energy of binding of the transition state to the enzyme
forms Glu
, Gly
(wild-type)
Glu
, Ser
(single mutant).
G
=
-RTln((k
/K
)
/(k
/K
)
). b-g, the energy of interaction of each side chain with
the transition state (Kcal/mol) in the hydrolytic reaction.
Calculations shown are from k
/K
terms (units
are s
mM
) obtained for
all the substrates tested.
The kinetics of the purified enzymes, with ampicillin as substrate,
suggest that the individual mutations are independent (Fig. 2b). Thus, the loss in interaction energy of the
double mutant with the transition state is close to the algebraic sum
of the free energies of the single mutants. It is interesting to note
that the K value for the double mutant is mostly
accounted for by the mutation G238S (Table 3). If K
is a good measure of the affinity for the
substrate, then this result suggests that the second alteration E104M
has little effect on the conformation of the active site, at least for
the interaction with this substrate.
In contrast, with cefuroxime as
substrate, the catalytic properties of the mutants are accounted for
only by the modification E104M (Table 2). When the change G238S
is added, in the context of the single mutant E104M, no additional
effect is observed (Fig. 2c). Unexpectedly, the
favorable effect shown by the E104M single mutant in the wild-type
context is even higher when this alteration is added in the G238S
context. The gain in interaction energy observed for the double mutant
contrasts with the lower value expected from independent single
mutants. The analysis of the K values of the
single and double mutants with this substrate reveals that the binding
of this substrate with the active site is very similar to that of the
wild type. This result is in contrast with the proposed structural
effect for the mutation G238S (Huletsky et al., 1993) and
observed in this work with the penicillin substrates tested and with
cephaloridine. This fact, and others (see below), point to the
difficulties in predicting the effect of some mutations on the
interaction with different substrates.
With other substrates, the
analysis of the individual contributions of the single mutants is more
complicated. With some substrates (6-aminopenicillanic acid,
cephaloridine, and benzylpenicillin), a relatively independent behavior
of the single mutations is observed (Fig. 2, d-f). The loss in interaction energy of the double
mutant with the transition state deviates little from the value
expected from independent effects of each mutation. With
benzylpenicillin as a substrate, the results show that there are no
effects of adding the modification E104M in the G238S context (Fig. 2f). This result might suggest more severe
alterations (as sensed by the substrate benzylpenicillin) of the active
site in response to the modification G238S. With all the above
substrates, there are additional variations in K shown by the single mutants as compared with the double mutant (Table 3).
With cefotaxime as substrate neither single mutant
significantly alters the K when compared with the
reported wild-type value. However, there is a decrease in the K
in the double mutant. The analysis of the
individual contributions, using k
/K
values, shows that the gain in interaction energy of the double
mutant is a little more than the value expected from independent
mutants (Fig. 2g). It is also clear from Fig. 2g that adding either mutation in the context of
the other produces an identical and more favorable improvement in the k
/K
parameter as compared
with the effect of the single mutations in the wild-type context. This
seems to indicate some kind of interdependence of both mutations that
might explain the small increase in interaction energy shown by the
double mutant.
Based on the lowered K for most
-lactams, Huletsky et al.(1993) suggested that the single
mutation G238S has a structural role. Our results show a similar trend.
However, the present study contributes some significant additional
information, namely the G238S mutation does not lower K
against second and third generation cephalosporins as shown by
the results obtained with the antibiotics cefuroxime and cefotaxime.
Interestingly, the double mutant E104M/G238S also shows a decrease in K
for cefotaxime, in spite of the fact that the
single substitutions have no effect on K
. With
cefuroxime, the K
of the double mutant is the same
as that observed with the single variants. The G238S variant shows
drastically reduced k
values against all substrates
tested except cefotaxime, where a distinct increase was observed. With
this latter substrate it is clear that the ability of the single
mutants to hydrolyze it is due to an improvement in the catalytic
parameters, but the combination of both single mutants results in an
improvement of the binding parameter as well. The ability to catalyze
hydrolysis of the second generation cephalosporin cefuroxime is
provided entirely by the modification E104M. In contrast, the change
G238S is detrimental in the wild-type context decreasing the k
and k
/K
parameters against this same substrate. Strikingly, the single
variant E104M and the double mutant show the same improvement in the
ability to hydrolyze cefuroxime.
The data obtained for the E104M
variant are more difficult to interpret in structural terms. Sowek et al.(1991) suggested that the hydrolysis of ceftazidime by
the variant E104K could be explained by an interaction between the new
positive charge of the Lys and the carboxylate of the
aminothiazole oxime side chain of the substrate. However, because the
same variant presented an enhanced activity against substrates with no
carboxyl group on the side chain, such as cefotaxime, the authors
proposed the formation of new favorable hydrogen bond interactions
between the side chain of Lys
and the substrate side
chain. With our variant, because the long hydrophobic side chain of
M104 cannot form hydrogen bonds, it is clear that alternative
explanations are necessary in order to understand the better hydrolytic
capabilities of this mutant with all the substrates tested when
compared both with the wild-type enzyme and with the E104K variant (see
Table VI of Sowek et al.(1991)). As can be seen in Fig. 3, Glu
of TEM
-lactamase (Strynadka et al., 1992; coordinates kindly provided by Dr. M. N. G.
James) could be playing a role in the replenishment of the hydrolytic
water molecule in a similar way as proposed for Asn
in
the Bacillus licheniformis
-lactamase (Knox and Moews,
1991). As shown in Fig. 3, Asn
may be able to
interact with water molecules 565 and 667. Strikingly, the substitution
Met for Glu in position 104 produces a better enzyme than the wild type
against all substrates tested. This results might suggest a lesser
importance of the outer rim in the replenishment mechanism as compared
with Asn
in the central position of the observed train of
water molecules. Alternatively, the substitution E104M could provide a
hydrophobic character to the active site making more favorable
interactions with the hydrophobic tip of the
-lactam side chain
substituent as suggested for the Staphylococcus aureus enzyme
(Herzberg and Moult, 1987). The fact that K
is not
altered in the interaction of the E104M mutant against all substrates
tested suggests that the hydrophobic interactions stabilize
preferentially the transition state as compared with the ground state
of the substrate. The modeling results of the mutants E104M and
E104M/G238S with benzylpenicillin docked in the active site show that
the calculated interaction distances between the
-lactam carbonyl
carbon atom and the oxygen of Ser
and the distances
between the
-lactam carbonyl and the oxyanion hole are closer to
the calculated wild-type values than those shown by the less active
mutant G238S (Table 5).
Figure 3:
Stereoview of some active site residues
(TEM coordinates kindly provided by Dr. M.N.G. James). Some
crystallographic water molecules are included as solid
spheres. The potential water molecules contacting residue 104 are
indicated. Penicillin G (BPE) is positioned for -face
acylation by reactive Ser
as modeled by Moews et
al.(1990).
In summary, our results show that the
disturbance of a complex set of interactions between different residues
and the substrate, probably together with subtle conformational
changes, can result in unpredictable specificity alterations. The
specificity changes could be the result of the improvement of binding
interactions in some regions of the active site (such as the
modification G238S) or in the improvement of the transition state
stabilization due to alterations in other region of the active site
(such as the modification E104M). Our results add to a data base that
should prove useful once structural data of these mutants is obtained.
This, together with detailed computer simulations may help clarify the
molecular basis of -lactamase substrate interaction.