Site-directed mutagenesis of beta-lactamase I: role of Glu-166.

Two Glu-166 mutants of beta-lactamase I from Bacillus cereus 569/H were constructed: one with a lengthened side chain (E166Cmc, the S-carboxymethylcysteine mutant) and the other with the side chain shortened and made non-polar (E166A). Their kinetic properties were studied and compared with those of the wild-type and the E166D mutant (with a shortened side chain) previously made by Gibson, Christensen and Waley (1990) (Biochem. J. 272, 613-619). Surprisingly, with good penicillin substrates, Km, kcat. and kcat./Km of the two conservative mutants (E166Cmc and E166D) are similar to those of the non-conservative mutant E166A. Their kcat. values are 3000-fold lower than that of the wild-type enzyme, showing that Glu-166 is a very important residue. The acylenzyme intermediate of E166A and a good substrate, penicillin V, was trapped by acid-quench and observed by electrospray ionization mass spectrometry, suggesting that Glu-166 is more important in catalysing the deacylation step than the acylation step. The beta-lactamase I E166A mutant is about 200-fold more active than the Bacillus licheniformis E166A mutant with nitrocefin or 6 beta-furylacryloyl-amidopenicillanic acid as substrate. This suggested that other groups in the active site of the beta-lactamase I mutant may activate the catalytic water molecule for deacylation.

The kinetics of slow-binding and slow, tight-binding inhibition: the effects of substrate depletion.

Inhibitors with dissociation constants in the micromolar to nanomolar range are important, but hard to characterize kinetically, especially when the substrate concentration in the assay is less than Km. When inhibition increases during the course of the assay (slow-binding inhibition) the concentration of substrate may decrease appreciably. Methods that take substrate depletion into account are described for analysing experiments in which the initial substrate concentration is below Km. Fitting progress curves gives the rate constants for the second (slow) step in a two-step mechanism. An approximate value for the overall dissociation constant may be determined from measurements of rates when the reaction is treated as a first-order process. When the concentrations of inhibitor and enzyme are comparable numerical methods are required. Procedures, suitable for implementation on a microcomputer, for the solution of the differential equations and the fitting of progress curves are described.

Site-directed mutagenesis and substrate-induced inactivation of beta-lactamase I.

The substrate-induced inactivation of beta-lactamase I from Bacillus cereus 569/H has been studied. Both the wild-type enzyme and mutants have been used. The kinetics follow a branched pathway of the type recently analysed [Waley (1991) Biochem. J. 279, 87-94]. The substrate cloxacillin (a penicillin) formed an acyl-enzyme (characterized by m.s.), and it was probably the instability of this intermediate that brought about inactivation. A disulphide bond was introduced into beta-lactamase I (the wild-type enzyme lacks this bond) by site-directed mutagenesis: Ala-77 and Ala-123 were replaced by cysteine. Spontaneous oxidation yielded the disulphide. The activity of this newly cross-linked enzyme was a little diminished, but the stability towards inactivation by cloxacillin was not increased. A second mutant of beta-lactamase I was studied: this mutant lacked the first 17 residues, i.e. the first alpha-helix. The mutant had reduced activity towards ordinary (non-inactivating) substrates and no hydrolysis of cloxacillin could be detected. These mutant enzymes were expressed in Bacillus subtilis, and were purified from the extracellular medium.

An easy method for deriving steady-state rate equations.

The scope and limitations of a simple and satisfactory method of deducing steady-state rate equations is described. This method (called the Flux Method) consists in writing down the flux in successive steps of the reaction, and calculating the relative concentration of enzyme forms and thence the turnover time. Kinetic mechanisms for linear and branched pathways are used as examples of this method.

Characterization of cell-bound papain-soluble beta-lactamases in BRO-1 and BRO-2 producing strains of Moraxella (Branhamella) catarrhalis and Moraxella nonliquefaciens.

In Moraxella (Branhamella) catarrhalis and Moraxella nonliquefaciens strains isolated from clinical specimens in the south of Sweden two variants of beta-lactamase were distinguished by isoelectric focusing (IEF). The BRO-1 (Ravasio type) enzyme was the most common in Branhamella catarrhalis, constituting about 90% of the beta-lactamase found in this species, while the BRO-2 enzyme (1908 type) was as common as BRO-1 in Moraxella nonliquefaciens. The determinants mediating the production of BRO-1 and BRO-2 were both transferable by conjugation. Cell-bound beta-lactamase from reference strains producing BRO-1 and BRO-2 could be solubilized by papain digestion. The isoelectric point of the solubilized enzymes differed distinctly between BRO-1 (pI 6.5) and BRO-2 (pI 6.9). The molecular species of BRO-1 and BRO-2 released by papain digestion were purified by affinity chromatography with phenylboronic acid agarose gel. They had identical molecular weights of approximately 28,000. Their kinetic constants were indistinguishable for a number of substrates and beta-lactamase inhibitors.

Crystalline enzyme kinetics: activity of the Streptomyces R61 D-alanyl-D-alanine peptidase.

The specificity constant, kcat/Km, for the hydrolysis of hippuryl-mercaptoacetate by crystals of the Streptomyces R61 D-D peptidase was measured by reaction of the thiol produced with 4,4'-dithiodipyridine. The values of kcat/Km for the crystal and in solution were the same (within experimental error). A novel method for treating the lag in the progress curves was developed.

The kinetics of substrate-induced inactivation.

The kinetics of a branched-pathway mechanism for a simple enzymic reaction were studied. In this mechanism there is reversible formation of an inactive form of the second complex along the pathway. This substrate-induced inactivation typically results in the progress curve showing a burst. Three parameters can be obtained from the progress curve: the initial rate, the final rate and the rate constant characterizing the transient. The rate constant for the conversion of the inactive form of the complex into the active form can be obtained either from these parameters or by measuring the regain of enzymic activity. The partition ratio can also be obtained from the three parameters; this is the ratio of the rate of conversion of complex into product to the rate of conversion of complex into inactive form. Simulations give guidance to the conditions required for accurate determinations of the rate constants.

Use of electrospray mass spectrometry to directly observe an acyl enzyme intermediate in beta-lactamase catalysis.

Electrospray mass spectrometry was used to directly observe intact acyl enzyme complexes formed between a class C beta-lactamase (from Enterobacter cloacae P99) and four poor substrates/inhibitors. In each case the molecular weight difference between the unreacted and the reacted beta-lactamase was consistent with the formation of an acyl enzyme.

Site-directed mutagenesis of beta-lactamase I. Single and double mutants of Glu-166 and Lys-73.

Two single mutants and the corresponding double mutant of beta-lactamase I from Bacillus cereus 569/H were constructed and their kinetics investigated. The mutants have Lys-73 replaced by arginine (K73R), or Glu-166 replaced by aspartic acid (E166D), or both (K73R + E166D). All four rate constants in the acyl-enzyme mechanism were determined for the E166D mutant by the methods described by Christensen, Martin & Waley [(1990) Biochem. J. 266, 853-861]. Both the rate constants for acylation and deacylation for the hydrolysis of benzylpenicillin were decreased about 2000-fold in this mutant. In the K73R mutant, and in the double mutant, the rate constants for acylation were decreased about 100-fold and 10,000-fold respectively. All three mutants also had lowered values for the rate constants for the formation and dissociation of the non-covalent enzyme-substrate complex. The specificities of the mutants did not differ greatly from those of wild-type beta-lactamase, but the hydrolysis of cephalosporin C by the K73R mutant gave 'burst' kinetics.

Beta-lactamases as fully efficient enzymes. Determination of all the rate constants in the acyl-enzyme mechanism.

The rate constants for both acylation and deacylation of beta-lactamase PC1 from Staphylococcus aureus and the RTEM beta-lactamase from Escherichia coli were determined by the acid-quench method [Martin & Waley (1988) Biochem. J. 254, 923-925] with several good substrates, and, for a wider range of substrates, of beta-lactamase I from Bacillus cereus. The values of the acylation and deacylation rate constants for benzylpenicillin were approximately the same (i.e. differing by no more than 2-fold) for each enzyme. The variation of kcat./Km for benzylpenicillin with the viscosity of the medium was used to obtain values for all four rate constants in the acyl-enzyme mechanism for all three enzymes. The reaction is partly diffusion-controlled, and the rate constant for the dissociation of the enzyme-substrate complex has approximately the same value as the rate constants for acylation and deacylation. Thus all three first-order rate constants have comparable values. Here there is no single rate-determining step for beta-lactamase action. This is taken to be a sign of a fully efficient enzyme.

Some uses of extrapolation in kinetics.

Extrapolation procedures are shown to be useful for obtaining kinetic parameters from irreversible enzymic reactions in which there are two intermediates, under both single-turnover and steady-state conditions. Small excesses of one component are treated as if they were large excesses, which is convenient in practice. The method has also been applied to a non-enzymic reversible bimolecular reaction.

Kinetic characterization of the acyl-enzyme mechanism for beta-lactamase I.

beta-Lactamase I catalyses the hydrolysis of penicillins by an acyl-enzyme mechanism. A procedure was developed for determining the rate constants for the acylation and deacylation steps for the good substrates benzylpenicillin and phenoxymethylpenicillin; this depends on determining the fraction of enzyme that is present as acyl-enzyme in the steady state.

Integrated 2:2 and 3:3 rate equations of enzyme kinetics.

Simple Michaelis-Menten kinetics give an equation for the initial rate, and the integrated version describes progress curves for experiments when the only reason for the rate's declining is the depletion of substrate. The integrated versions of the more complicated 2:2 and 3:3 rate equations are now presented.

Imipenem as substrate and inhibitor of beta-lactamases.

The interaction between imipenem, a carbapenem antibiotic, and two representative beta-lactamases has been studied. The first enzyme was beta-lactamase I, a class-A beta-lactamase from Bacillus cereus; imipenem behaved as a slow substrate (kcat. 6.7 min-1, Km 0.4 mM at 30 degrees C and at pH 7) that reacted by a branched pathway. There was transient formation of an altered species formed in a reversible reaction; this species was probably an acyl-enzyme in a slightly altered, but considerably more labile, conformation. The kinetics of the reaction were investigated by measuring both the concentration of the substrate and the activity of the enzyme, which fell and then rose again more slowly. The second enzyme was the chromosomal class-C beta-lactamase from Pseudomonas aeruginosa; imipenem was a substrate with a low kcat. (0.8 min-1) and a low Km (0.7 microM). Possible implications for the clinical use of imipenem are considered.

Beta-lactamase inhibitors. The inhibition of serine beta-lactamases by specific boronic acids.

Many beta-lactamases have active-site serine residues, and are competitively inhibited by boronic acids. Hitherto, the boronic acids used have lacked any structural resemblance to the substrates of beta-lactamases. Phenylacetamidomethaneboronic acid, trifluoroacetamidomethaneboronic acid and 2,6-dimethoxybenzamidomethaneboronic acid have now been synthesized. The first of these contains the side-chain moiety of penicillin G, and the last that of methicillin. The pH-dependence of binding of the first inhibitor to beta-lactamase I from Bacillus cereus revealed pK values of 4.7 and 8.2 for (presumably) active-site groups in the enzyme. The kinetics of inhibition were studied by cryoenzymology and by stopped-flow spectrophotometry. These techniques provided evidence for a two-step mechanism of binding of the first two boronic acids mentioned above to beta-lactamase I, and for benzeneboronic acid to a beta-lactamase from Pseudomonas aeruginosa. The slower step is probably associated with a change in enzyme conformation as well as the formation of an O-B bond between the active-site serine hydroxy group and the boronic acid.

beta-lactamase I from Bacillus cereus. Structure and site-directed mutagenesis.

The sequence of the gene for beta-lactamase I from Bacillus cereus 569/H has been redetermined. Oligonucleotide-directed mutagenesis has been carried out, and the effects of the changes on the ampicillin-resistance of Escherichia coli TG1 expressing the mutant genes have been studied. Lysine-73, close to the active-site serine-70 and a highly-conserved residue, has been converted into arginine. This change had a large effect on activity, but did not abolish it. An even larger effect was found in the mutant in which glutamate-166 had been converted into glutamine; this had little or no activity. On the other hand, the conversion of glutamate-168 into aspartate gave fully active enzyme. Glutamate-166 is an invariant residue, but glutamate-168 is not. Alanine-123 has been replaced by cysteine, to give active enzyme; this change forms part of the plan to introduce a disulphide bond into the enzyme.

An X-ray-crystallographic study of beta-lactamase II from Bacillus cereus at 0.35 nm resolution.

Crystals of beta-lactamase II (EC 3.5.2.6., 'penicillinase') from Bacillus cereus were grown with Cd(II) in place of the natural Zn(II) cofactor and stabilized by cross-linking with glutaraldehyde. Their space group is C2, the cell dimensions are a = 5.44 nm, b = 6.38 nm, c = 7.09 nm and beta = 93.6 degrees, and there is one molecule in the asymmetric unit. Diffraction data were collected from cross-linked crystals of the Cd(II)-enzyme, the apoenzyme and six heavy-atom derivatives. The electron-density map calculated at 0.35 nm resolution reveals the essential Cd(II) ion surrounded by three histidine residues and one cysteine residue. The position of a glutamic acid residue, modification of which destroys activity [Little, Emanuel, Gagnon & Waley (1986) Biochem. J. 233, 465-469], suggests the probable location of the active site of the enzyme. Two minor Cd(II) sites not essential for activity were also located. The structure of the apoenzyme at this resolution appears to differ from that of the Cd(II)-enzyme only in the orientation of two of the histidine residues and the cysteine residue that surround the metal ion.