Mechanism of inhibition of the class C beta-lactamase of Enterobacter cloacae P99 by cyclic acyl phosph(on)ates: Rescue by return

As previously described (Pratt, R.F.; Hammar, N.J.J. Am. Chem. Soc. 1998, 1 20, 3004.), 1-hydroxy-4,5-benzo-2,6-dioxaphosphorinone(3)-1-oxide (salicylo yl cyclic phosphate) inactivates the class C beta -lactamase of Enterobacte r cloacae P99 in a covalent fashion. The inactivated enzyme slowly reverts to the active form. This paper shows that reactivation involves a recycliza tion reaction that regenerates salicyloyl cyclic phosphate rather than hydr olysis of the covalent intermediate. The inactivation, therefore, is a slow ly reversible covalent modification of the active site. The thermodynamic d issociation constant of the inhibitor from the inactivated enzyme is 0.16 m uM. Treatment of the inactivated enzyme with alkali does not produce salicy lic acid but does, after subsequent acid hydrolysis, yield one molar equiva lent of lysinoalanine. This result proves that salicyloyl cyclic phosphate inactivates the enzyme by (slowly reversible) phosphorylation of the active site serine residue. This result contrasts sharply with the behavior of ac yclic acyl phosphates which transiently inactivate the P99 beta -lactamase by acylation (Li, N.; Pratt, R.F. J. Am. Chem. Soc. 1998, 120, 4264.). This chemoselectivity difference is explored by means of molecular modeling. Ra ther counterintuitively, in view of the relative susceptibility of phosphat es and phosphonates to nucleophilic attack at phosphorus, 1-hydroxy-4,5-ben zo-2-oxaphosphorinanone(3)-1-oxide, the phosphonate analogue of salicyloyl cyclic phosphate, did not appear to inactivate the P99 beta -lactamase in a time-dependent fashion. It was found, however, to act as a fast reversible inhibitor (K-i = 10 muM). A closer examination of the kinetics of inhibiti on revealed that both on and off rates (9.8 x 10(3) s(-1) M-1 and 0.098 s(- 1), respectively) were much slower than expected for noncovalent binding. T his result strongly indicates that the, inhibition reaction of the phosphon ate also involves phosphylation of the active site. Hence, unlike the situa tion with bacterial DD-peptidases covalently inactivated by beta -lactams, the P99 beta -lactamase inactivated by the above cyclic acyl phosph(on)ates can be rescued by return. Elimination of the recyclization reaction would lead to more effective inhibitors.