Kinetic mechanism of metallo-beta-lactamase L1 from Stenotrophomonas maltophilia

The reaction of nitrocefin with metallo-beta-lactamase L1 from Stenotrophom onas maltophilia was studied using rapid-scan and stopped-flow ultraviolet- visible (UV-vis) studies in an effort to discern the kinetic mechanism used by L1 to hydrolyze penicillins and cephalosporins. Rapid-scan and stopped- flow UV-vis studies of nitrocefin hydrolysis by L1 identified three species : (1) the substrate (nitrocefin) displayed an absorbance peak at 390 nm (ep silon = 11 500 M-1 cm(-1)) that decreased during the reaction with a rate c onstant of 170 +/- 30 s(-1) (2) the product (hydrolyzed nitrocefin) display ed an absorbance peak at 485 nm (epsilon = 17 420 M-1 cm(-1)) that increase d during the reaction with rate constant of 40 +/- 1 s(-1); and (3) an inte rmediate displayed an absorbance peak at 665 nm (epsilon = 32 000 M-1 cm(-1 )) that increased initially with a rate constant of 190 +/- 3 s(-1) and the n decreased with a rate constant of 38 +/- 2 s(-1). Single-turnover experim ents demonstrated that there were no pre-steady-state bursts in the reactio n of L1 with nitrocefin; moreover, the progress curves could be fit to a ki netic mechanism that includes the formation of a transient intermediate by using KINSIM and the rate constants given above. Progress curves from exper iments conducted at different reaction conditions or with a different subst rate could also be fit to the proposed kinetic mechanism. The evidence for the presence of an intermediate along with kinetic simulations supports a h ydrolytic mechanism for L1 that involves an intermediate whose breakdown is rate-determining.