Fluorometric Method for Estimating the Kinetic Parameters of β-Naphthyl Triphosphate and ATP Hvdrolvsis by Acto-Heavy Meromyosin

Abstract

We have established a method to estimate the values of various kinetic parameters of actoheavy meromyosin (acto-HMM) ATPase, using a fluorescent ATP analog, β-naphthyl triphosphate (β-NapP₃); from the fluorescence intensity change accompanying β-NapP₃ hydrolysis, the various kinetic parameters of β-NapP₃ hydrolysis, including its product inhibition, were obtained. β-NapP₃ hydrolysis is inhibited competitively by ATP, resulting in different time courses of fluorescence intensity change in the presence and absence of ATP. From this difference, the values of kinetic parameters of ATP hydrolysis, including its product inhibition, can be estimated. By extending this method to the acto-HMM system, seventeen parameters in a reaction scheme for the concurrent hydrolysis of ATP and β-NapP₃, including association constants between F-actin and substrate-free or substrate-bound HMM, were obtained. The kinetic parameters estimated for ATP hydrolysis were in good agreement with those in the literature.