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Serine beta-lactamases contribute widely to the beta-lactam resistance phenomena. Unfortunately, the intimate details of their catalytic mechanism remain elusive and subject to some controversy even though many "natural" and "artificial" mutants of these different enzymes have been isolated. This paper is essentially focused on class C beta-lactamases, which contain a Tyr (Tyr150) as the first residue of the second conserved element, in contrast to their class A counterparts, in which a Ser is found in the corresponding position. We have modified this Tyr residue by site-directed mutagenesis. On the basis of the three-dimensional structure of the Enterobacter cloacae P99 enzyme, it seemed that residues Glu272 and His314 might also be important. They were similarly substituted. The modified enzymes were isolated and their catalytic properties determined. Our results indicated that His314 was not required for catalysis and that Glu272 did not play an important role in acylation but was involved to a small extent in the deacylation process. Conversely, Tyr150 was confirmed to be central for catalysis, at least with the best substrates. On the basis of a comparison of data obtained for several class C enzyme mutants and in agreement with recent structural data, we propose that the phenolate anion of Tyr150, in conjunction with the alkyl ammonium of Lys315, acts as the general base responsible for the activation of the active-site Ser64 during the acylation step and for the subsequent activation of a water molecule in the deacylation process. The evolution of the important superfamily of penicillin-recognizing enzymes is further discussed in the light of this proposed mechanism.

Bacillus subtilis DppA is a binuclear zinc-dependent, D-specific aminopeptidase. The X-ray structure of the enzyme has been determined at 2.4 A resolution by a three-wavelength MAD experiment. The structure reveals that DppA is a new example of a 'self-compartmentalizing protease', a family of proteolytic complexes. Proteasomes are the most extensively studied representatives of this family. The DppA enzyme is composed of identical 30 kDa subunits organized in a decamer with 52 point-group symmetry. A 20 A wide channel runs through the complex, giving access to a central chamber holding the active sites. The structure shows DppA to be a prototype of a new family of metalloaminopeptidases characterized by the SXDXEG key sequence.