Kinetic and structural characterization of reversibly inactivated beta-lactamase.

The reversible inhibition of beta-lactamase I from Bacillus cereus by cloxacillin, methicillin, and nafcillin has been systematically investigated. For these substrates the enzymatic reaction involves partitioning of the substrate between turnover and inhibition. Typically, concentrations of several hundred millimolar are necessary for complete inactivation. The completely inactivated enzyme could be formed by incubation at temperatures above 20 degrees C, where inhibition competes more effectively with turnover, and then stabilized by dropping the temperature to 0 degrees C or lower. The inactivated enzyme was rapidly separated from unreacted substrate and product at low temperature by centrifugal gel filtration or ion exchange and examined by far-UV circular dichroism for evidence of a conformational change. At pH 7 the inactivated enzyme had a secondary structure essentially identical with that of the native enzyme. The fluorescence emission spectrum of the inactivated enzyme (at pH 7) was also identical with that of the native enzyme. However, the inactivated enzyme was found to be considerably more sensitive to thermal denaturation, to acid-induced conformational isomerization, and to trypsinolysis than the native enzyme. We conclude from the circular dichroism results that the structure of the reversibly inactivated enzyme cannot be significantly different from that of the native enzyme. Therefore, previous findings that have been interpreted as indicating a major conformational change must be reevaluated. From examination of the low-resolution crystallographic structure of the enzyme we propose that the most likely cause of the inactivation is an alternate conformational state of the acyl-enzyme intermediate involving movement of one or more of the alpha-helices forming part of the active site. Such a structural effect could leave the secondary structure unchanged but have significant effects on the tertiary structure, catalysis, mobility, and susceptibility to trypsin and denaturation. We propose that the underlying physical reason why certain beta-lactam substrates bring about this "substrate-induced deactivation", or suicide inactivation, of the enzyme is due to the presence of the alternative acyl-enzyme conformation of similar free energy to the productive one, in which one (or more) essential catalytic group is no longer optimally oriented for catalyzing deacylation. Thus for substrates with a slow rate of deacylation (less than or equal to 100 s-1) the conformational transition can compete effectively on the time scale of the turnover reaction.