First-order Kinetics in the Study of Enzymes: Applications to the Reporter Substrate Method and to the Estimation of kcatKm

Abstract

The study of the interactions between enzymes and inactivators can often be performed with the help of the reporter substrate method in which the time-dependent decrease of the rate of substrate disappearance (or product formation) is monitored. In the present contribution, we wish to describe examples of the utilization of this rapid and efficient method for reactions whose rates can be monitored by spectrophotometric or fluorimetric measurements. After the collection of the data in an Excel file, a very simple program can be applied to extract the values of pseudo-- first-order rate constants. The inactivation can be complete or result in a steady-state if the inactivated adduct is not totally stable or if the inactivation reaction is reversible. Similarly, the method can be used in the cases of so-called “slow binding” inhibitors. The same type of analysis allows the easy determination of kcat/Km values for substrates for which the Km value is rather low. We show that this very rapid method (less than 5 min) yields very good values of the desired kinetic parameter even if the total absorbance variations are very low (0.1 or less). In conclusion, the described experimental approach is particularly useful when applied to the reporter substrate method but it also allows the estimation of the kcat/Km parameter even if the Km value is rather low. The authors wish to dedicate this paper to the memory of the late Michel Rinné (1941-2009) whose contribution to making the data analysis program very user-friendly was invaluable.