Mechanism of reaction of acyl phosph(on)ates with the beta-lactamase of Enterobacter cloacae P99

A series of acyl phosph(on)ates has been prepared to more closely examine t he details of the interactions of this class of molecule with beta -lactama ses. In general, they were found to react with the class C beta -lactamase of Enterobacter cloacae P99 in two ways, by acylation and by phosphylation. The acyl-enzymes generated by the former reaction were transiently stable with half-lives of between 3 and 45 s, of comparable lifetime therefore to those generated by the inhibitory beta -lactams cefotaxime, cefuroxime, and cefoxitin. On the other hand, phosphylation led to a completely inactive e nzyme. In general, the second-order rate constants for acylation (k(cat)/K- m) were larger than for phosphylation (k(i)). As expected on chemical groun ds, phosphylation was found to be relatively mere effective for the phospho nates than the phosphates. The acyl phosphates were much more effective acy lating agents however. The acylation reaction was found to be enhanced by h ydrophobic substituents in both the acyl and leaving group moieties. Thus, the most reactive compound in this series was benzo[b]thiophene-2-carbonyl 2'-naphthyl phosphate with a K-m value of 0.15 muM and a k(cat) of 0.2 s(-1 ); k(cat)/K-m is therefore 1.3 x 10(6) s(-1) M-1 making this compound the m ost specific acyclic acylation reagent for this beta -lactamase yet describ ed. Significant substrate inhibition by this compound suggested that furthe r binding regions may be available for exploitation in inhibitor design. A linear free energy analysis showed that the transition states for acylation of the beta -lactamase by aroyl phosphates are analogues of the correspond ing aryl boronic acid adducts. Molecular modeling suggested that the aroyl phosphates react with the P95, beta -lactamase with the aroyl group in the side chain/acyl group site of normal substrates and the phosphate in the le aving group site. In this orientation, the phosphate leaving group interact s strongly with Lys 315.