Methanolysis of nitrocefin catalyzed by one and two Zn(2+) ions. A simplified model for class B beta-lactamases

The methanolysis of nitrocefin (1) was investigated at 25 degrees C in anhydrous methanol as a function of [Zn(2+)] and as a model for the chemistry believed to occur in the active site of Zn(2+)(-)beta-lactamases. In the absence of metal ion, the vs rate constant profile shows a long plateau region having k(obs) = 9 x 10(-5) s(-1) between 7.5 and 12 generated after ionization of the COOH of 1 (kinetic pK(a) of 7.34) followed by attack of CH(3)O(-) with k(CH)3(O) = 1.18 M(-1) s(-1). Strong catalysis of the methanolysis is observed at all pH values between 7.95 and 11.34 in the presence of as little as 0.05-3 mM Zn(2+). Plots of the pseudo-first-order rate constant for methanolysis (k(obs)) as a function of reveal a saturation phenomenon indicative of formation of a 1:Zn(2+) complex, followed by a linear section indicative of the intervention of a second Zn(2+) ion in promoting the methanolysis of the complex. The two processes can be separated since the slope of the linear part of the plots gives the second-order rate constant (k(2)(obs)) for the second Zn process, while the intercept gives the spontaneous rate constant for methanolysis of 1:Zn(2+) (k(cat.)(obs)). A plot of log k(cat.)(obs) vs is bell-shaped, maximizing at 10, with ascending and descending domains both first order in [CH(3)O(-)]. A plot of log k(2) vs gives an ascending domain second order in [CH(3)O(-)], followed by a plateau above 9.5. The data are analyzed in terms of a one-Zn and two-Zn model in which the former involves rate-limiting attack of methoxide on a 1:Zn(2+) complex up to the maximum and a spontaneous reaction of 1:Zn(2+):(CH(3)O(-))(2), while the latter involves bimolecular attack of Zn(2+)(CH(3)O(-))(2) on both 1:Zn(2+) and 1:Zn(2+):(CH(3)O(-))(2). The relevance of these observations is discussed in terms of the currently accepted mechanism for hydrolyses of beta-lactams promoted by the Zn(2+)(-)beta-lactamases.