On incubation of the chlorinated 6-spiroepoxypenicillin anilides (I) and (II) [formula: see text] with beta-lactamase 1 from Bacillus cereus, three distinct processes are observed. The inhibitors act as (a) substrates, the turnover of which respectively results in a single product, namely 6-substituted 2(H)-3,4-dihydro-1,4-thiazine, (b) a transiently inhibited enzyme complex, and finally (c) an irreversibly inactivated enzyme complex. Although differing only in their stereochemistry at one centre, the anilide (K) is a more potent irreversible inactivator of beta-lactamase I than is compound (II). Analysis of irreversibly inactivated beta-lactamase I by isoelectric focusing and inspection of peptide fragmentation maps indicated that irreversible inactivation appears to be accompanied by covalent modification. These studies reveal that the chlorinated 6-spiroepoxypenicillin anilide (I) is a mechanism-based beta-lactamase inhibitor.
