(Received for publication, October 11, 1996)
From the § Centre d'Ingénierie des Protéines and Laboratoire d'Enzymologie, Université de Liège, Institut de Chimie B6, Sart-Tilman, B-4000 Liège, Belgium
The catalytic pathway of class A -lactamases
involves an acyl-enzyme intermediate where the substrate is
ester-linked to the Ser-70 residue. Glu-166 and Lys-73 have been
proposed as candidates for the role of general base in the activation
of the serine OH group. The replacement of Glu-166 by an asparagine in
the TEM-1 and by a histidine in the Streptomyces albus G
-lactamases yielded enzymes forming stable acyl-enzymes with
-lactam antibiotics. Although acylation of the modified proteins by
benzylpenicillin remained relatively fast, it was significantly
impaired when compared to that observed with the wild-type enzyme.
Moreover, the E166N substitution resulted in a spectacular modification
of the substrate profile much larger than that described for other
mutations of
-loop residues. Molecular modeling studies indicate
that the displacement of the catalytic water molecule can be related to this observation. These results confirm the crucial roles of Glu-166 and of the "catalytic" water molecule in both the acylation and the
deacylation processes.
DD-peptidases and most -lactamases, belong to the superfamily
of active-site serine penicillin-recognizing enzymes (1). The
interaction between these proteins and
-lactams involves the
formation of an acyl-enzyme (E-S*) intermediate
where the antibiotic is covalently bound to the active-site serine
residue,
![]() |
(Eq. 1) |
The mechanism by which serine -lactamases hydrolyze penicillins and
cephalosporins has received a lot of attention and, for the class A
enzymes the identity of the residue involved in the activation of the
active serine (Ser-70 in the ABL numbering system (8)) has been subject
of controversy. Both Lys-73 (9) and Glu-166 (10) have been proposed as
potential candidates for this essential role. By contrast, the function
of Glu-166 in activating the hydrolytic water molecule during the
deacylation step is unanimously recognized. According to Adachi
et al. (11) and Strynadka et al. (9),
accumulation of an acyl-enzyme during the interaction between the TEM-1
Glu-166
Asn mutant (E166N) and benzylpenicillin suggested that the
mutation affected only the deacylation step in a severe manner. In
contrast, kinetic studies of the Glu-166
Ala mutant of the
Bacillus licheniformis
-lactamase (12) and of the Glu-166
Asp mutant of the Bacillus cereus I
-lactamase (13)
showed that accumulation of the acyl-enzyme could result from
simultaneous but different decreases of the kinetic parameters characterizing the acylation and deacylation steps.
The present paper reports the analysis of the kinetic properties of the
TEM-1 E166N mutant. Methods were developed to monitor the rapid
formation of the acyl-enzyme. One of the most striking results was a
sharp modification of the substrate profile of the enzyme. To explain
these results, the interactions between different -lactams and the
E166N mutant were analyzed by molecular modeling. These confirmed the
role of the catalytic water molecule in the mechanism and underlined
the importance of the
-loop residues in the specificity profile.
Similar kinetic data were obtained with the E166H mutant of the
Streptomyces albus G class A
-lactamase.
Strains and Plasmids
Escherichia coli TG1 strain ((lac-pro), Sup E,
thi, hsdD5/F
traD36, proA
B+,
lacIq, lacZ
M15) was used for routine transformation, DNA
preparation and production of the TEM-1
-lactamase and as a host
strain for M13 phage growth. The Streptomyces lividans TK24
strain (14) was utilized for the production of the Streptomyces
albus G
-lactamase.
Plasmid pAD25 (15) encodes both the tetracycline resistance and an
isopropyl -D-thiogalactopyranoside-inducible TEM-1
-lactamase. It was used both for the mutagenesis procedure and the
expression of the WT1 and mutant
enzymes.
The gene encoding the S. albus G -lactamase was cloned
into the M13tg131 phage for mutagenesis. The mutant gene was entirely sequenced, recloned into the pIJ702
Streptomyces plasmid (a
gift from Dr. J. Altenbüchner, Universität-Regensburg,
Germany), and expressed in S. lividans.
Nucleic Acid Techniques
Sequencing was performed by the method of Sanger et al. (16) using the Sequenase sequencing kit (U. S. Biochemical Corp.). T4 DNA ligase and kinase were purchased from Boehringer Mannheim GmbH. Oligonucleotides were purchased from Eurogentec (Liège, Belgium). Specific Streptomyces DNA manipulations, such as protoplast preparation, transformation, and plasmid extraction, were based on Hopwood et al. (17).
Mutagenesis
The E166N TEM-1 -lactamase mutant was obtained by inverse
polymerase chain reaction mutagenesis (18) with the following oligonucleotides: 5
-AAGGCGAGTTACATGATCCCC-3
;
5
-GTAAGTCGAGGCC
A
GGTTGCTAG-3
. The latter carried the two mismatched bases designed to mutate the GAA
(Glu) codon into AAC (Asn).
Amplification was performed with the Vent DNA polymerase (Biolabs) in the buffer supplied by the manufacturer to which MgSO4 was added to a final concentration of 4 mM; 30 cycles (60 s at 94 °C, 60 s at 56 °C, 270 s at 72 °C) were performed on the reaction mixture. The polymerase chain reaction product was subsequently treated as described by Imai et al. (19) and used to transform E. coli TG1.
For the E166H mutant of the S. albus G -lactamase,
mutagenesis was performed as described by Eckstein et al.
(20). The oligonucleotide used to replace the GAC (Glu) codon by CAC
(His) was 5
-CTCGACCGCTGG
ACCCGGAGCTGAAC-3
.
Chemicals
Benzylpenicillin was from Rhône Poulenc (Paris, France), cefuroxime and ceftazidime were from Glaxo Group Research (Greenford, Middlesex, United Kingdom), cefotaxime from Hoechst-Roussel (Romainville, France). Imipenem, moxalactam, temocillin, and cefoxitin from Merck, Sharp and Dohme Research Laboratories (Rahway, NJ). Cephalosporin C and cephaloridine from Eli Lilly and Co. (Indianapolis, IN). All these compounds were kindly given by the respective companies. [14C]Benzylpenicillin (54 mCi/mmol) was purchased from Amersham International.
Isopropyl -D-thiogalactopyranoside and tetracycline were
purchased from Sigma. Fluoresceyl-6-aminopenicillanic acid was prepared as described by Lakaye et al. (21).
Expression and Purification of Mutant -Lactamases
The E166H mutant of the S. albus G -lactamase was
isolated from 3 liters of culture supernatant and purified as described by Brannigan et al. (22) with an additional chromatographic step on a Superdex 75 HR 10/30 column in 25 mM sodium
phosphate buffer, pH 7.
For the production of the E166N TEM-1 mutant, E. coli TG1
was grown at 30 °C in 15 liters of M9 minimal medium supplemented with 50 µg/ml tetracycline. When the A550
value reached 0.6, 0.1 mM isopropyl
-D-thiogalactopyranoside was added and the culture was
further incubated for 3 h. Cells were collected, suspended in a 30 mM Tris-HCl buffer, pH 7, containing 27% sucrose (w/v) and
lysed by lysozyme (1 mg/ml final) as described by Dubus et al. (15). Cell debris were discarded by centrifugation and the supernatant dialyzed against 10 mM Tris-HCl, pH 7.5. The
enzyme was purified as described by Vanhove et al. (23). The
fractions containing the enzyme were identified by submitting samples
of fluoresceyl-6-aminopenicillanic acid-labeled enzyme to
electrophoresis on a 15% polyacrylamide gel in the presence of SDS
(24).
The purity of the different enzyme preparations was verified by Coomassie Blue staining of SDS-polyacrylamide electrophoresis gels. The TEM-1 mutant was titrated with [14C]benzylpenicillin and found to be more than 95% pure. The E166H mutant of S. albus G was only 50% pure and remained contaminated by a single, low molecular mass protein which did not interfere with the kinetic measurements.
Determination of the Kinetic Parameters
Due to the very low k3 values observed
with these two mutants the determination of their kinetic parameters
rested on methods similar to those used for penicillin-binding proteins
rather than for regular -lactamases. Unless otherwise mentioned, all
the kinetic assays were performed at 30 °C in 50 mM
sodium phosphate buffer, pH 7.
Determination of k2/K Values
Pseudo-first order reactions are only obtained when the
-lactam concentration is at least five times greater than that of enzyme. Due to the high k2/K values,
these conditions where only fulfilled when the experiments were
performed in a stopped-flow apparatus. The
-lactam concentration was
also decreased close to that of enzyme and analysis performed according
to the general second order equation. Finally after the direct
determination of the k2/K values for
cefoxitin and cephalosporin C, those for the other compounds were
determined by a competition method.
Direct Determination with Manual Mixing (Method A)
The enzyme solution was added with cefoxitin, cephalosporin C,
cefotaxime, moxalactam, or ceftazidime. The total volume was 0.5 ml and
the final enzyme concentration 16 µM. The antibiotic concentration was always slightly higher than that of the enzyme. Decrease of the A260 value resulting from the
opening of the -lactam ring was monitored with the help of an Uvikon
spectrophotometer connected to a COPAM PC88C microcomputer. The data
were fitted to Equation 2 using the Enzfitter program (Biosoft,
Cambridge, United Kingdom (25)) yielding the value of the second-order rate constant k, i.e.
k2/K
, where K
= (k
1 + k2)/k+1. Under conditions
of similar initial enzyme and substrate concentration, the general
Equation 2 prevails.
![]() |
(Eq. 2) |
Under our experimental conditions (S0 > E0) concentrations could be replaced by absorbance values as in Equation 3, leading to Equation 4,
![]() |
(Eq. 3) |
![]() |
(Eq. 4) |
![]() |
Stopped-flow Experiments (Method B)
The enzyme (16 µM) was mixed with increasing concentrations of cefoxitin with the help of a Biologic Stopped-flow apparatus (Grenoble, France). The S0/E0 ratio ranged from 1.2 to 25. For data analysis, Equation 2 was adapted to the stopped-flow recording conditions. Alternatively, for the highest S0/E0 ratios the pseudo-first order approximation was used, yielding identical results.
Competition Method (Method C)
The enzyme (0.9 µM) was added with a mixture of
[14C]benzylpenicillin (B) and another antibiotic (I) at
concentrations ranging from 2.5 × 106 to 1.0 × 10
4 M. After 5 min, the reaction was stopped
and the protein precipitated, isolated, and the radioactivity
determined as described by Martin and Waley (26). Since both antibiotic
concentrations were larger than that of the enzyme and the incubation
time was much shorter than the half-life of both acyl-enzymes (see
below), the respective quantities of labeled
(E-B* formed with
[14C]benzylpenicillin) and unlabeled
(E-I* formed with the other antibiotic)
acyl-enzymes were given by Equation 5 as shown by Frère et
al. (27),
![]() |
(Eq. 5) |
Direct Binding of a Labeled Antibiotic (Method D)
With the E166H mutant of the S. albus G enzyme the
second-order rate constant for acylation
(k2/K) was obtained by monitoring acyl-enzyme formation as a function of time (method D). The enzyme was
incubated with an excess of [14C]benzylpenicillin
(S0
E0) and after increasing
periods of time, the reaction was stopped by addition of 1% SDS. The
labeled acyl-enzyme was isolated by SDS-polyacrylamide gel
electrophoresis and quantified by fluorography (24).
Determination of k3
Labeled AntibioticThe protein (1.6 µM) was
reacted with an excess of [14C]benzylpenicillin
(104 M) and the excess of free antibiotic
eliminated by addition of 600 ng of the wild-type enzyme. The mixture
was incubated at 30 °C and after increasing periods of time, the
residual acyl-enzyme quantified after precipitation as above (26).
After formation of the acyl-enzyme in
50 mM cacodylate buffer, pH 7, at 30 °C, the excess of
unlabeled antibiotic was hydrolyzed by addition of the Zn--lactamase
of B. cereus (1 µM) and 100 µM
ZnCl2. The acyl-enzyme was then left to decay during
different time periods. 1 mM EDTA was added to inactivate
the Zn-
-lactamase and the regenerated active-serine enzyme was
quantified by saturation with [14C]benzylpenicillin and
precipitation as above. With both labeled and unlabeled antibiotics,
the degradation of the acyl-enzyme obeyed simple first-order kinetics
(28).
pH Dependence of the Enzyme Activity
The following pH range was explored: acetic acid/sodium acetate, pH 4-5.5, sodium phosphate, pH 6-8, Tris-HCl, pH 8.5-9. All the buffers were 50 mM. These experiments were performed with the help of method A for the E166N mutant and method D for the E166H mutant. For the TEM-1 wild-type enzyme, the kcat/Km value was obtained by determining initial rates (v0) at substrate concentrations well below the Km value.
The data obtained for the wild-type TEM-1 and the E166N mutant were fitted to Equation 6 by means of the Enzfitter program,
![]() |
(Eq. 6) |
Thermal Denaturation Curves
Temperature-induced unfolding was monitored by recording the intrinsic fluorescence of the TEM-1 E166N as described by Vanhove et al. (29).
The cuvette containing the sample (1.4 ml) was heated at a rate of 0.9 °C/min using a thermostatically regulated circulating water bath. The enzyme unfolded reversibly in a simple co-operative fashion. Unfolding curves were analyzed according to Equation 7 by means of the nonlinear regression data analysis Enzfitter program,
![]() |
(Eq. 7) |
Proteolysis Experiments
A mixture containing 0.2 mg of enzyme and 0.02 mg of trypsin was incubated in 100 mM Tris-HCl, pH 7.5, at 40 °C. Aliquots were removed and trypsin was inactivated by addition of 40 mg/ml soybean trypsin inhibitor (Sigma). Quantification of the mutant residual activity was performed by titration with [14C]benzylpenicillin.
Optimized Structures of the Substrates
The structures of the -lactam molecules were optimized by the
AM1 semi-empirical method (Dewar et al. (30)). CNDO charges were adopted for these ligands. The bond lengths, bond angles, and ring
dihedral angles of the antibiotics were constrained to the AM1
values.
Molecular Modeling
Models of the mutant enzymes were obtained from the x-ray structures of the wild-type proteins (5) and the local conformational space of the mutated amino acid was searched by a minimum perturbation approach (31).
The system was solvated by cubes of standard "Monte Carlo" water molecules and the positions of these molecules after minimization were compared to those in the crystal structures. The geometry of the protein was optimized with the AMBER 4.1 set of programs (32) using a distance-dependent dielectric constant.
The -lactam molecules were docked in the modeled active sites with
the
-lactam amide oxygen atom oriented into the oxyanion-hole formed
by the main chain nitrogen atoms of Ser-70 and Ala-237. Hydrogen bonds
appeared to be formed between the C7 side chain amide group and the
N
2 amide nitrogen atom of Asn-132 and the main chain
oxygen atom of Ala-237. The energy of the Michaelis complexes thus
obtained was minimized using AMBER (32).
About 16 mg of the TEM-1 E166N mutant were obtained per liter of culture. The protein was purified to 95% homogeneity with a yield of 45%. For the S. albus G mutant, the culture and purification yields were 20 mg/liter and 25%, respectively; as stated above, the final preparation was only 50% pure. Attempts to eliminate the low molecular mass contaminant by various chromatographic procedures, including molecular sieve filtration, remained unsuccessful.
Kinetic PropertiesThe values of the rate constants obtained for the TEM-1 E166N mutant are reported in Table I. As expected and in agreement with the observations of Adachi et al. (11) and Strynadka et al. (9), the rates of deacylation (k3) were exceedingly low.
|
Different strategies were utilized to determine the second-order rate
constant (k2/K), characteristic of
the acylation rate. Rapid acylation could not be monitored by the usual
methods. With [14C]benzylpenicillin, the reaction was so
rapid that it could not be followed by manual mixing methods. In
consequence, the first experiments were performed with compounds
exhibiting lower acylation rates and whose modifications upon
acyl-enzyme formation could be directly monitored by spectrophotometric
methods. The manual method A allowed the determination of the
k2/K
values for cephalosporin C,
cefoxitin, cefotaxime, and ceftazidime. That Equation 2 applied was
confirmed by the fact that similar
k2/K
values were obtained at
cefoxitin concentrations ranging from 20 to 40 µM. The
stopped-flow method, which required much larger quantities of proteins
was only utilized with cefoxitin. Here the antibiotic concentrations were as high as 400 µM. An average
k2/K
value of 2200 ± 440 M
1 s
1 was found, in fair
agreement with that obtained in the manual mixing experiments
(4500 ± 130 M
1 s
1). The
values for the other compounds were obtained by the competition method
C. First, competitions between [14C]benzylpenicillin and
cephalosporin C or cefoxitin were performed, allowing the computation
of the k2/K
value for the
14C-labeled compound. Subsequently, the values for all the
other
-lactams were derived from competition experiments with
[14C]benzylpenicillin.
For good substrates of the wild-type enzyme (benzylpenicillin and cephaloridine), the E166N mutation resulted in a decrease of the acylation rates by 2-3 orders of magnitude. This was, however, much less drastic than for the k3 value. The most striking results concerned the modification of the substrate profile.
In contrast to what occurred with the good substrates, the
k2/K for the poorest WT substrates
(i.e. temocillin, cefoxitin, and moxalactam which bear a
methoxy side chain on the
-face of the
-lactam ring) were
increased by several orders of magnitude. With the oxyiminocephems, the
k2/K
value was not much affected. In
consequence the k2/K
values of the
mutant only spanned 3 (from 400 to 400 × 103)
versus 7 orders of magnitude for the wild-type protein.
With benzylpenicillin and the S. albus G E166H mutant,
similar results were obtained, involving a significant decrease of k2/K accompanied by a much more
drastic decrease of k3 (Table II).
|
The pH dependence of
the acylation rate of the E166N mutant by cefoxitin was studied using
method A (see Fig. 1). The pH dependence of the
k2/K value could be fitted to
Equation 6 with pK1 and pK2 values of 6.0 and 8.5, respectively. With
the wild-type enzyme pK values of 7.2 and 9 were found. The
mutation thus resulted in a shift of the curve to lower pH values, with
a more significant effect to the acid limb. The
k3 constant was not significantly influenced by
the pH value (not shown).
The k2/K value of the E166H mutant
of the S. albus G
-lactamase (Table II) exhibited a new
pH dependence which seemed to indicate that the base form of the
His-166 side chain was adequately oriented to actively participate in
the acylation step. By contrast, the k3 value at
pH 5 was higher than at pH 7 or 9. This suggested that the His-166
residue of the mutant enzyme was not involved in the deacylation step
as a general base catalyst.
The melting temperature
(Tm) and the enthalpy (Hm) of
unfolding obtained by fitting the experimental curves to Equation 7
were: 51.1 ± 0.2 °C and 112 ± 7 kcal/mol °C, 43 ± 1.4 °C and 87 ± 5 kcal/mol °C for the wild-type and the
E166N mutant proteins, respectively. This result highlighted a
significant destabilization of the mutant which was corroborated by the
proteolysis experiments in which the pseudo-first order rate constants
(ki) characterizing trypsin-mediated inactivation
were 0.29 min
1 and 0.012 min
1 for the TEM-1
E166N and WT proteins (33), respectively.
The strong hydrogen bonds found
between the carboxylic group of Glu-166 and the side chain nitrogen
atoms of Lys-73 and Asn-170 of TEM-1 disappeared in the E166N mutant,
allowing free rotation of these residues. In the most stable structure
obtained by conformational analysis, the Asn-170 acyl group was rotated
by 180° around the C-C
bond with
respect to the orientation found in the WT enzyme. A similar rotation
was observed in the structure of the E166A mutant of the B. licheniformis
-lactamase (34). There was no change in the
position of Lys-73 relative to its WT position. All calculations were
performed considering both the protonated and unprotonated states of
Lys-73 and yielded similar results.
The position of the "catalytic" water molecule in the mutant was
found to be the same whether it was generated by a Monte Carlo
simulation or minimized from the position found in the WT structure.
The water hydrogen atoms were oriented toward the Asn-166 and Asn-170
Os, respectively. By comparison with the WT structure, it was shifted by 1.3 Å away from Ser-70 with which it no longer formed a H-bond (Fig. 2).
In the E166H mutant, the plane of the imidazole side chain was rotated
by 75° with respect to that formed by the glutamate carboxylate in
the WT enzyme. The catalytic water molecule slightly (0.8 Å) moved to
the bottom of the cavity away from its initial position and was thus
further from Ser-70. The lengths of the hydrogen bonds were 3.12 and
2.85 Å with an unprotonated and protonated Lys-73 residue,
respectively, as previously shown in the model of the E166D mutant of
the S. albus G enzyme (35). The N170 side chain was more
outward-oriented due to a 30° rotation around the
C-C
bond.
Three cephalosporins: cephaloridine, cefuroxime, and
cefoxitin, respectively, good, bad, and very bad substrates of the WT TEM-1 but of similar properties with the mutant were chosen as models
for the study of the specificity of the E166N mutant. Benzylpenicillin was also studied (not shown), yielding results similar to those obtained with cephaloridine (Fig. 3A).
The optimized structures of the Michaelis complexes were not very different from those found in the case of the WT enzyme. In all the complexes, the catalytic water molecule remained positioned as in the free mutant (Fig. 3B), so that it was not perturbed by the C7-methoxy group of cefoxitin, a situation very different from that observed with the WT enzyme. With cefuroxime the main additional observation was a decrease of the steric hindrance between the oxyimino group and the N170 side chain (Fig. 3C). Deprotonation of Lys-73 only resulted in a slight increase of the distance between the water and the Ser-70.
Interaction of the E166H Mutant with BenzylpenicillinThe
optimized Henri-Michaelis complex was not very different from that
computed for the wild-type enzyme (Fig. 4). The major difference was that the NH-CO bond of the C-6 substituent of penicillin was now closer to the N170 side chain due to the displacement of the
latter (about 3 Å).
Both mutant proteins were more poorly produced than the WT
enzymes. This might be related to the relative instability of the mutant proteins which increases their sensitivity to proteases as shown
for the TEM-1 E166N mutant. With this protein, utilization of a minimal
medium and a lower temperature significantly improved the production
yield. This instability might due to an increased mobility of the
-loop as shown for various other TEM-1 mutants (36, 37).
On the basis of a preliminary study of the TEM-1 E166N mutant, Adachi
et al. (11) concluded that the deacylation rate was severely
decreased while the acylation reaction was unimpaired. Since the first
conclusion was in disagreement with the data of Gibson et
al. (13) and Leung et al. (38), however, obtained with
different Glu-166 mutants of another enzyme, a careful kinetic study of
the TEM-1 E166N protein was performed. As expected and with all
substrates, our results indicated extremely large decreases of the
k3 value, a 109 factor for
benzylpenicillin. This result underlined the pivotal role of the
Glu-166 in the deacylation step, a feature on which a general consensus
has been reached. By contrast, with the good substrates, the
k2/K value, which characterizes the
efficiency of acylation and corresponds to the
kcat/Km of the WT enzyme, was
decreased by 2 or 3 orders of magnitude indicating a non-negligible,
although less pivotal role of Glu-166 in the acylation step. This was,
however, not true for all substrates since the acylation rates by
poorer substrates were unchanged or even slightly increased and for the
very bad substrates, cefoxitin and other compounds exhibiting a methoxy
group on the
-face, acylation was significantly facilitated.
Consequently, the "activity" profile of the enzyme was deeply
modified, with a spectacular leveling effect. According to Strynadka et al. (9), the position of the -loop was not strongly
modified in the mutant. The situation was the same with the B. licheniformis E166A mutant but quite different with the TEM-1
E166Y (39) and S. aureus D179N (40) proteins where the loop
was disordered. Here the most striking structural modifications were
the displacement of the water molecule W1 away from the active site
Ser-70 side chain and the disappearance of several strong hydrogen
bonds. It should be noted that this water molecule has been
hypothesized to serve as a relay in the activation of the Ser-70
hydroxyl group by the Glu-166 side chain acting as a general base in
the acylation process. However, an alternative mechanism has been
proposed by Strydnaka et al. (9), where the general base
would be the unprotonated side chain of Lys-73 and the Glu-166
carboxylate in the acylation and deacylation steps, respectively.
Although it would explain the properties of E166X mutants
which would catalyze the acylation reaction with the same efficiency as
the wild-type enzyme but would fail to deacylate, this hypothesis also
implies a significantly decreased pKa value for the
alkylammonium group of the lysine 73 side chain in the wild-type
enzyme. A recent NMR titration of the Lys-73 residue in the TEM-1
protein failed to supply evidence for such unusual
pKa value (41). Other Glu-166 mutants have been
prepared with other enzymes (12, 38, 42). Whenever detailed kinetic
studies were performed, both acylation and deacylation rates appeared
to be decreased by the mutation, although deacylation was sometimes
much more severely impaired. Moreover, the K73R mutant of the B. cereus 569/H
-lactamase I (13) was significantly more active
than its E166D counterpart. The drastic substrate profile modifications
observed in this present study suggest that the acylation of the mutant
might rely on an alternative mechanism. One can hypothesize that, as
proposed by Lamotte-Brasseur et al. (10) and Matagne
et al. (43), the very low acylation rates of the WT enzyme
by cefoxitin, moxalactam, and temocillin are due to a displacement of
W1 by the methoxy side chain of the substrates. With the mutant, the
Lys-73 side chain might replace Glu-166 as the general base as in
acetoacetate decarboxylase (44) and as suggested by Strynadka et
al. (9) for the WT
-lactamase. The disappearance of the
negative Glu-166 charge would explain a decrease of the Lys-73
pKa when compared to the WT enzyme.
This alternative acylation mechanism would be significantly less efficient for good substrates but, since it would not involve W1, the reaction rates with cefoxitin and similar compounds would be increased. Finally, the unchanged acylation rates with oxyiminocephalosporins would result from the same negative factor as with the good substrates compensated by a positive effect due to the increased mobility of the omega loop as observed for example with the Arg-164 mutants of the TEM-1 enzyme (37).
Note that an unprotonated Lys-73 may not be necessary to act as a
proton relay. The softness of the electronic atmosphere of the nitrogen
atom allows an easy adaptation to environmental modifications such as
the binding of a ligand. It is finally interesting to note that the
acylation rates observed with the E166N mutant are of the same order of
magnitude as those measured with several penicillin-binding proteins.
The Actinomadura R39 (45) DD-peptidase and B. licheniformis PBP1 (46) contain all the residues constituting the
conserved elements of the class A -lactamases with the exception of
the
-loop and, in consequence, have no equivalent of Glu-166 as
shown by the three-dimensional structures of the homologous Streptococcus pneumoniae PBP2x (47) and
Streptomyces K15
DD-peptidase.2
The results obtained with the S. albus G enzyme
generally support these conclusions. In this case, the
k3 value is also strongly decreased and this
residue does not appear to act as a general base in the deacylation
reaction, as indicated by the absence of significant variation of
k3 between pH 5 and 9. By contrast, the
k2/K value significantly increases
with increasing pH values and, although a real titration curve is not
observed, this might suggest that the base form of this residue might
participate in the acylation reaction.
These results underline a major role of the W1-Glu-166 pair in the
enzymatic acylation-deacylation process and indicate that although
Glu-166 is involved in both acylation and deacylation reactions in
class A -lactamases, these two reactions are not necessarily
"mirror" images of each other since the disappearance of Glu-166
affects the two steps in a different way. A careful kinetic analysis of
mutant enzymes is thus a prerequisite to meaningful mechanistic
conclusions.
We thank Dr. X. Raquet for many fruitful discussions.