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An amino acid residue functioning as a general base has been proposed to assist in the hydrolysis of beta-lactam antibiotics by the zinc-containing Bacillus cereus beta-lactamase II [Bicknell & Waley (1985) Biochemistry 24, 6876-6887]. Oligonucleotide-directed mutagenesis of cloned Bacillus cereus 5/B/6 beta-lactamase II was used in an 'in vivo' study to investigate the role of carboxy-group-containing amino acids near the active site of the enzyme. Substitution of asparagine for the wild-type aspartic acid residue at position 81 resulted in fully functional enzyme. An aspartic acid residue at position 90 is essential for beta-lactamase II to confer any detectable ampicillin and cephalosporin C resistance to Escherichia coli. Conversion of Asp90 into Asn90 or Glu90 lead to the synthesis of inactive enzyme, suggesting that the spatial position of the beta-carboxy group of Asp90 is critical for enzyme function.

The classification by structure allots beta-lactamases to (at present) three classes, A, B and C. The pH-dependence of the kinetic parameters for class B and class C have been determined. They differ from each other and from class A beta-lactamases. The class B enzyme was beta-lactamase II from Bacillus cereus 569/H/9. The plots of kcat against pH for the hydrolysis of benzylpenicillin by Zn(II)-requiring beta-lactamase II and Co(II)-requiring beta-lactamase II were not symmetrical, but those of kcat/Km were. A similar feature was observed for the hydrolysis of both benzylpenicillin and cephalosporin C by a class C beta-lactamase from Pseudomonas aeruginosa. The results have been interpreted by a scheme in which two ionic forms of an intermediate can give product, but do so at differing rates.

The 1H n.m.r. spectra of beta-lactamase II in the presence of Co(II) were studied. Analysis of the spectra suggests that Co(II) binds at the same two metal-binding sites as does Zn(II). The binding of Co(II) at the first site is much weaker than the binding of Zn(II) at this site, whereas the binding of Co(II) at the second site is tighter than the binding of Zn(II). The binding of Co(II) to the mono-zinc(II)-enzyme caused only one marked change in the spectrum, namely a decrease in the intensity of the resonances assigned to the C-2 and C-4 protons of one histidine residue (residue E). However, when the spectra of the apoenzyme and the Co(II)-enzyme were compared, there were many differences. A significant fraction of the protons in the whole molecule are affected by the binding of Co(II) at the first metal-ion-binding site (where the ligands are the enzyme's sole thiol group and three histidine residues). This may be because the first site is internal, or because of a difference in conformation between the apoenzyme and the mono-Co(II)-enzyme. The second site may be located on the surface of the molecule.

1. Four histidine-containing peptides have been isolated from a tryptic digest of the Zn2+-requiring beta-lactamase II from Bacillus cereus. One of these peptides probably contains two histidine residues. 2. The presence of one equivalent of Zn2+ substantially decreases the rate of exchange of the C-2 proton in at least two and probably three of the histidine residues of these peptides for solvent 3H. 3. It is concluded that peptides containing at least two of the three histidine residues acting as Zn2+ ligands at the tighter Zn2+-binding site of beta-lactamase II have been identified.

Beta-Lactamase II from Bacillus cereus was readily inactivated by incubation at pH 4.75 with a water-soluble carbodiimide plus a suitable nucleophile. In the early stages of the reaction, 1 equivalent of nucleophile was incorporated/equivalent of enzyme, whereas during the later stages a second equivalent of nucleophile was also incorporated. This latter process correlated with the blocking of the enzyme's single thiol group. Enzyme inactivated in the presence of the coloured nucleophile N-(2,4-dinitrophenyl)ethylenediamine was fragmented by pepsin digestion, and coloured peptides were isolated by gel filtration and h.p.l.c. Two major peptides, representing 52% of the incorporated label, were isolated and sequenced. Both peptides contained the incorporated label on glutamic acid-37, and it is concluded that this latter residue represents a catalytically essential carboxylic residue in beta-lactamase II.

The production and purification of a tetrameric zinc beta-lactamase from Pseudomonas maltophilia IID 1275 were greatly improved. Three charge variants were isolated by chromatofocusing. The subunits each contain two atomic proportions of zinc and (in two of the variants) one residue of cysteine. The thiol group is not required for activity, nor does it appear to bind to the metal. Replacement of zinc by cobalt, cadmium or nickel takes place at a measurable rate, and gives enzymes that are less active than the zinc enzyme. The properties of this enzyme differ from those of the other known zinc beta-lactamase, beta-lactamase II from Bacillus cereus. The amino acid sequence of the N-terminal 32 residues was determined; there is no similarity to the N-terminal sequences of other beta-lactamases.

An 8-thionocephalosporin was shown to be a substrate of the beta-lactamase II of Bacillus cereus, a zinc metalloenzyme. Although it is a poorer substrate, as judged by the Kcat./Km parameter, than the corresponding 8-oxocephalosporin, the discrimination against sulphur decreased when the bivalent metal ion in the enzyme active site was varied in the order Mn2+ (the manganese enzyme catalysed the hydrolysis of the oxo compound but not that of the thiono compound), Zn2+, Co2+ and Cd2+. This result is taken as evidence for kinetically significant direct contact between the active-site metal ion of beta-lactamase II and the beta-lactam carbonyl heteroatom. No evidence was obtained, however, for accumulation of an intermediate with such co-ordination present.