FIGURE 2.
Representative time course of enzymatic activity measurements to determine the association rate constants. The plots of the Mobs (defined as [E]t/([E]t + [B]0 − [E]0)) of the experimentally measured instantaneous enzymatic activity [E]t versus time (t) after mixing the TEM-1 (0.5 nm, top left), Bla1 (5 nm, top right), KPC-2 (1 nm, bottom left), and CTXM-14 (1 nm, bottom right) with 3-fold higher concentrations of BLIP-II (wild type or the indicated mutant) as described under “Experimental Procedures.” The solid circles are the Mobs of the wild-type BLIP-II, and the solid triangles are the Mobs of the indicated mutant, whereas the solid curves are the fitting curves of the second order association kinetics according to Equation 1 (see “Experimental Procedures”) to determine the appropriate kon values (tabulated in Table 1). The experiments to determine the association rate constants were performed in at least triplicate, and all of the data sets were combined to obtain the rate constant with an associated standard error through nonlinear regression.