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Substitution of Asp for Asn at Position 132 in the Active Site of TEM β -Lactamase

1995; Elsevier BV; Volume: 270; Issue: 2 Linguagem: Inglês

10.1074/jbc.270.2.775

1083-351X

Joel Osuna, Héctor Viadiu, Anthony L. Fink, Xavier Soberón,

Bacterial Genetics and Biotechnology

Using a random, combinatorial scheme of mutagenesis directed against the conserved SDN region of TEM β-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 pK1 and pK2 for catalysis were decreased about 1 unit relative to the pK′s for the wild type. This effect was predominantly due to changes in Km . In contrast to the wild-type, the pH-rate profiles of the mutant showed that Km 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, kcat for the mutant showed a similar pH dependence as the wild type. With 6-aminopenicillanic acid, kcat for the mutant was greater than that for the wild type. The nature of the 104 side chain may affect the environment of Asp132; 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 Asp132 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. Using a random, combinatorial scheme of mutagenesis directed against the conserved SDN region of TEM β-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 pK1 and pK2 for catalysis were decreased about 1 unit relative to the pK′s for the wild type. This effect was predominantly due to changes in Km . In contrast to the wild-type, the pH-rate profiles of the mutant showed that Km 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, kcat for the mutant showed a similar pH dependence as the wild type. With 6-aminopenicillanic acid, kcat for the mutant was greater than that for the wild type. The nature of the 104 side chain may affect the environment of Asp132; 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 Asp132 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. INTRODUCTIONClass A β-lactamases are serine hydrolase members of a group of enzymes that destroy β-lactam antibiotics by hydrolyzing the sensitive four-membered β-lactam ring. TEM β-lactamase from Escherichia coli is the most common plasmid-mediated enzyme of this class. Crystal structures of the β-lactamases of Staphylococcus aureus (Herzberg, 1991), Bacillus licheniformis (Knox and Moews, 1991), and, recently, E. coli TEM β-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 β-lacta-mase is shown in, E+S⇀↽K0ES⇀k2EA⇀k2E+P(Eq. 1) where EA represents the acyl-enzyme. For such a scheme, Kcat=k2k3k2+k3(Eq. 1a) and Km=ksk3k2+k3(Eq. 1b) 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 Glu166 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 β-lactamase showed that Asn 132 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 TEMβ-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 132 residue and the substrate side chain.EXPERIMENTAL PROCEDURESMaterialsBenzylpenicillin, ampicillin, 6-APA 1The abbreviations used are: APA6-aminopenicillanic acidMICminimum inhibitory concentrationWTwild type. , and carbenicillin (Fig. 1) were purchased from Sigma. Nitrocefin was a gift from Glaxo. Taq polymerase was purchased from Promega. The polymerase chain reactions were performed in a Biosycler oven. Restriction enzymes, Klenow fragment, and ligase were obtained from Boehringer Mannheim.Mutagenesis of the SDN RegionMutant β-lactamases were made as described in Merino et al.(1992). Mutations were introduced via polymerase chain reaction and sequenced with Sequenase (U. S. Biochemical Corp.). The primer used for mutagenesis was 5′-CTGCCATAACCATGNNG/CNNG/CNNG/CACTGCGGCCAAC-3′, where N means an equimolar mixture of all four nucleotides and G/C means a mixture with 50% each G and C.Determination of the Minimum Inhibitory Concentration (MIC)The MICs were determined by a dilution method on Luria broth agar plates at 37°C, with inocula of 10-50 colony-forming units dispensed in 5-μl drops. The plates contained the antibiotic at several different concentrations plus kanamycin at 25 μg/ml. The minimum concentration inhibiting the growth of cells was taken as the MIC.Purification ProcedureThe wild type and mutant enzymes were purified as follows. β-Lactamase (WT or N132D) was expressed and purified from E. coli. Fresh colonies were inoculated into 6 liters of Luria-Bertani media and grown on a 37°C shaker for 12-14 h. After a cold shock treatment, cell debris was removed by centrifugation (6000 rpm for 10 min) and the cell-free supernatant mixed with of the total volume of 0.02 M sodium acetate, pH 4.8. Some proteins were removed by precipitation by gently swirling on a shaker at 4°C; the precipitate was removed by centrifugation as above and the supernatant (containing β-lactamase) was batch-loaded onto a CM-Sepharose (CL-6B from Sigma) column previously pre-equilibrated with the same sodium acetate buffer. The nonbound proteins were washed with the same buffer for several hours and the bound proteins eluted with a linear gradient of 0-0.4 M NaCl. Fractions containing β-lactamase activity were pooled and exchanged via ultrafiltration with an Amicon YM10 filter into 0.025 M imidazole buffer, pH 7.0. This protein solution was loaded onto a chromatofocusing column (PBE94 from Pharmacia) using the same buffer as start buffer and eluted with a pH gradient of 7 to 4 using the pH 4 Polybuffer 74-HCl from Pharmacia. Fractions containing β-lactamase activity were pooled and exchanged into 0.05 M potassium phosphate buffer, pH 7.0, by ultrafiltration as above. All proteins were purified to more than 90% homogeneity as determined by SDS-polyacrylamide gel electrophoresis and isoelectrofocusing-polyacrylamide gel electrophoresis using the Pharmacia PhastSystem.KineticsThe concentration of β-lactamase was determined by using an ε280 = 29,400 M -1 cm -1 . pH rate profiles for benzylpenicillin, ampicillin, and 6-APA were determined from complete progress curves, using substrate concentrations at least 5 × Km or from initial velocities measurements, and the data fitted to the Michaelis-Menten equation. The data were analyzed with Kaleidagraph (Abelbeck software) on a Macintosh computer. All points were determined at least in triplicate. The carbenicillin pH profile was obtained only by initial velocities using 10-15 different substrate concentrations bracketing the Km and the data fitted to the Michaelis-Menten equations using the Kaleidagraph program. The nitrocefin data were obtained by initial velocities measured at the respective optimal pH for the wild-type and N132D mutant. Because the Km for nitrocefin was quite high, only kcat /Km could be determined, using measurements of the first-order rate of hydrolysis under conditions of substrate concentrations much less than Km. Substrate hydrolysis was measured at 30°C with a Perkin-Elmer 320 spectrophotometer according to the method of Waley(1974). All buffers contained 0.5 M KCl and were brought to a final concentration of 50 mM buffer. The buffers used for the pH profiles were sodium acetate for pH 4.0-5.5, potassium phosphate for pH 6.0-7.5, and Tris-HCl for pH 8.0-8.5. The wavelengths for monitoring substrates were 240 nm for penicillins and 482 nm for nitrocefin.Computational ProceduresThe active site of the wild-type or N132D TEM β-lactamases, defined by all the amino acid residues and water molecules present within 10 Å radius around the Ser 70 Cα, was optimized by energy minimization using the all-atom consistence valence force field parameters and charges. The Asp 132 residue in the N132D mutant was modeled both in the protonated (neutral) or ionized form. The initial binding interactions of benzylpenicillin and 6-aminopenicillanic acid with the enzymes were studied by manual docking of the substrate to the active site followed by unconstrained energy minimization of the final complex. The substrates were manually fitted into the active site to position the β-lactam carbonyl oxygen in the oxyanion hole and to maximize hydrogen bonding. The model substrate was adjusted manually to relieve obvious steric problems with the protein.RESULTSDescription of Mutant EnzymesRandom combinatorial mutagenesis directed against the SDN region and selective screenings in ampicillin plates produced the mutant N132D described in this work. In order to probe the effect of secondary alterations in the environment of the N132D mutant, we modified the Glu 104 residue to different amino acids, including Asn and Gln, some of which are present in other class A β-lactamases (Asn in B. licheniformis enzyme).Antibiotic SusceptibilityAntibiotic susceptibility testing of E. coli strain JM101 carrying the plasmid-borne gene for the wild-type or mutant enzymes is shown in Table 1. The MIC values show that the N132D mutant has greatly decreased resistance to ampicillin (MIC = 20 μg/ml) compared with the wild-type (MIC = >2560 μg/ml). All of the double mutants N132D/E104X were unable to confer the resistance phenotype, as shown by the MIC levels against ampicillin, 6-APA, and benzylpenicillin.Tabled 1Substrate and pH Activity ProfilesThe effect of the substitution of Asp for Asn at residue 132 on the kinetic parameters for several penicillin substrates (Fig. 1) are reported in Figure 2:, Figure 3:, Figure 4:. As can be seen in Fig. 2, kcat of the N132D mutant is 2-fold lower with all the penicillin substrates tested, except 6-APA, as compared with the wild-type values. The data from the corresponding kcat versus pH curves were fit to to obtain pK values. Kobs=klim1+H/K1+K2/H(Eq. 2) Figure 4:kcat /Km 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2:kcat pH profiles for the N132D mutant (open squares) and the wild-type (closed circles) β-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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The pKa values of the acidic limb (pK1) for kcat had values similar to those of the wild-type, and the pKas of the basic limb (pK2) were slightly displaced to lower values (Table 3 and Fig. 2).Tabled 1With 6-APA as substrate, the kcat of the mutant had higher values than the wild type. With this substrate, both pK1 and pK2 for the mutant enzyme had values very similar to the wild type (Table 3 and Fig. 2).With carbenicillin as a substrate, the wild-type kcat showed an anomalous pH dependence, similar to that shown for the Bacillus cereus β-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 β-lactamase. kcat=k(ESH2)limH/K1+1+K2/H+K2K3/H2 +k(ESH)limH2/K1K2+H/K2+1+K3/H(Eq. 3) The behavior of Km for the mutant can be separated in two groups, depending of the substrate used: (i) with benzylpenicillin, ampicillin, and carbenicillin as substrates, Km 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 Km of the mutant enzyme approaches wild-type values (Fig. 3, A-C). (ii) With 6APA as a substrate, the Km 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).Figure 3:Km pH profiles for the N132D mutant and the wild-type enzyme. The panels are the same as described in the legend to Fig. 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)When compared at the pH optima, the catalytic efficiency (kcat /Km) of the mutant was around 30-50% of the wild-type efficiency, depending on the substrate (Fig. 4 and 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 pK2 and 0.5 in pK1 for the N132D mutant relative to the values for the wild-type (Table 3). With carbenicillin as substrate, the pH dependence of kcat /Km 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 ().Tabled 1When tested with nitrocefin, an activated cephalosporin substrate, the mutant enzyme had a kcat /Km value around 2-3% of the wild-type enzyme when compared at the pH optima (kcat /KmWT = 4.2 × 106;mz -1 s -1 and kcat /Km mutant = 8.5 × 104; mz -1 s -1 ).Energy-minimized DockingAs shown in Table 4, the calculated interaction distances between the enzymes and the substrates correlate well with the observed pH activity behavior of the enzymes (Figure 2:, Figure 3:, Figure 4:). The energy-minimized dockings of both substrates to the mutant protein, modeled with a protonated Asp 132 residue, show that the calculated distances are similar to those shown by the wild type. These results are in agreement with the high activity shown by the mutant enzyme at low pH (around 5-5.5). The calculated distances obtained in the complex of benzylpenicillin with the mutant protein modeled with an ionized Asp 132 were significantly longer than the previous modeled complexes. However, the contact distances shown between 6-APA and this same modeled protein were similar to the calculated ones for the wild-type or mutant protein modeled with a protonated Asp 132 residue. These results are consistent with the pH stability shown by the Km values for this substrate. In the oxyanion hole, the hydrogen bond between the β-lactam carbonyl and the backbone NH237 is stronger than the one with the backbone NH70, a result similar to that obtained by Juteau et al.(1991) when modeling substrate binding by the ROB-1 enzyme.Tabled 1DISCUSSIONHerzberg and Moult(1987) and Moews et al.(1990) working with different class A β-lactamases have proposed that the amide group of the Asn 132 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 β-lactamase, have shown this same contact. Also, the side chain of Asn 132 forms a strong hydrogen bond with that of Lys 73 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 132 is in close contact with the side chain of the Asn 104 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 132 and the main chain carbonyl of Glu 104 or Ala 104 , 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 β-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 104 in the E. coli TEM β-lactamase), we superimposed the structurally homologous residues of S. aureus (Ala 104 ) and B. licheniformis 749/C (Asn 104 ) enzymes (coordinates 3BLM and 4BLM, respectively; Protein Data Bank, Brookhaven National Laboratory). The deacylating water is labeled 321. Penicillin G (BPE) is positioned for α-face acylation by reactive Ser 70 as modeled by Moews et al. (1990).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Jacob et al. (1990a, 1990b) working with S. albus β-lactamase variants, where Asn 132 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, kcat 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 132 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, kcat 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 kcat and the similarity, at this pH, of Km to wild-type values (Fig. 3, A-C). At pH higher than 5.5, Km is sharply increased, whereas in the wild-type enzyme Km 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 kcat 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 kcat /Km against pH in the mutant relative to the wild type (Fig. 4, A-C) is due mostly to the effect on the Ks component of Km.With a side chain-lacking substrate, such as 6-APA, both kcat and Km 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 (k3), which would result in an increase in kcat and Km, as observed. The behavior of Km for the N132D mutant with 6-APA is different when compared with that of the other substrates (Fig. 3D). Km 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 104 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 pKa. 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 132 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 73 is not seriously affected, and the effects proposed for the acylation step (k2) 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 (k3) 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 132 modeled in ionized form). The results suggest better contact distances between benzylpenicillin and the active site of the mutant with Asp 132 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 132 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 kcat 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 Km. 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 β-lactamase are more disruptive than the one present in our mutant. The loss of the contact with Lys 73 (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 132 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 132 with different groups of the active site, it is also indirectly involved in the catalytic mechanism. Mutations in Asn 132 could affect both the acylation rate by impairing the interaction of this residue with Lys 73 , and the deacylation rate by altering the control of the replenishment of the hydrolytic water as suggested by the contact of Asn 132 with a water molecule of a train leading from the solvent to the interior of the active site. INTRODUCTIONClass A β-lactamases are serine hydrolase members of a group of enzymes that destroy β-lactam antibiotics by hydrolyzing the sensitive four-membered β-lactam ring. TEM β-lactamase from Escherichia coli is the most common plasmid-mediated enzyme of this class. Crystal structures of the β-lactamases of Staphylococcus aureus (Herzberg, 1991), Bacillus licheniformis (Knox and Moews, 1991), and, recently, E. coli TEM β-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 β-lacta-mase is shown in, E+S⇀↽K0ES⇀k2EA⇀k2E+P(Eq. 1) where EA represents the acyl-enzyme. For such a scheme, Kcat=k2k3k2+k3(Eq. 1a) and Km=ksk3k2+k3(Eq. 1b) 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 Glu166 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 β-lactamase showed that Asn 132 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 TEMβ-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 132 residue and the substrate side chain.