Direct fluorimetric measurement on the hydrolysis of β-naphthyl triphosphate, a fluorescent ATP analog, by heavy meromyosin

Abstract

A fluorescent ATP analog, β-naphthyl triphosphate, was hydrolyzed to β-naphthyl diphosphate and orthophosphate by heavy meromyosin ATPase. In the process of hydrolysis the fluorescence intensity of β-naphthyl triphosphate changed remarkably. Thus, the rate of β-naphthyl triphosphate hydrolysis is evaluated directly and continuously by measuring the time course of fluorescence intensity. In the presence of Ca2+, the Michaelis constant (Km) of β-naphthyl triphosphate hydrolysis by heavy meromyosin was similar to that of ATP hydrolysis. While, in the presence of Mg2+ the Km of β-napthyl triphosphate hydrolysis was 9.0·10−6 M, much larger than the value of ATP hydrolysis, indicating that the apparent affinity of the enzyme for β-naphthyl triphosphate is less than that for ATP. The pH dependence of β-naphthyl triphosphatase activity resembled that of ATPase activity, suggesting a similarity in the mechanism of hydrolysis of the two substrates.