Automated analysis of enzyme inactivation phenomena: Application to β-lactamases and DD-peptidases

Abstract

In the presence of a reporter substrate, the progressive inactivation of an enzyme was easily studied by directly transmitting absorbance readings to a microcomputer. Pseudo-first order rate constants as high as 0.3 sec −1 were rapidly and accurately measured. When utilization of the reporter substrate did not exceed 10%, the rate of the reaction ( v t) could be considered as proportional to the active enzyme concentration at any time during the analysis and the decrease of v t, was first order with time. This simple method was used to follow the inactivation of β-lactamases (EC 3.5.2.6) by various physical and chemical agents. When a large proportion (30–80%) of reporter substrate was destroyed, a correction was introduced to account for the corresponding decrease of its rate of utilization. This enabled experiments to be performed with a DD-peptidase and a substrate exhibiting a low Δε upon hydrolysis. For the first time, the inactivation of a penicillin-sensitive enzyme by a β-lactam could be continuously and directly observed. Finally, the method was extended to the study of hysteresis phenomena.