Mechanism of Reaction of Acyl Phosph(on)ates with the β-Lactamase of Enterobacter cloacae P99

Abstract

A series of acyl phosph(on)ates has been prepared to more closely examine the details of the interactions of this class of molecule with beta-lactamases. 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 enzyme. In general, the second-order rate constants for acylation (k(cat)/K(m)) were larger than for phosphylation (k(i)). As expected on chemical grounds, phosphylation was found to be relatively more effective for the phosphonates than the phosphates. The acyl phosphates were much more effective acylating agents however. The acylation reaction was found to be enhanced by hydrophobic 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 microM 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 most specific acyclic acylation reagent for this beta-lactamase yet described. Significant substrate inhibition by this compound suggested that further 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 corresponding aryl boronic acid adducts. Molecular modeling suggested that the aroyl phosphates react with the P99 beta-lactamase with the aroyl group in the side chain/acyl group site of normal substrates and the phosphate in the leaving group site. In this orientation, the phosphate leaving group interacts strongly with Lys 315.