Heavy meromyosin Mg-ATPase: presteady-state and steady-state Hplus release.

Abstract

The presteady state and steady state release of protons during hydrolysis of ATP by myosin has been studied by using a proteolytic subfragment of myosin, heavy meromyosin, isolated from rabbit skeletal muscle. Under the conditions of 0.5 M KCl, 9 mM MgCl(2), pH 8, and 25 degrees , the rate of the H(+) initial burst is proportional to the concentration of ATP added, and shows no plateau even when the concentration of ATP added is increased up to 750 muM. The apparent second order rate constant is approximately 7 x 10(5) M(-1) sec(-1). When the concentration of ATP added is equal to or less than the concentration of heavy meromyosin catalytic sites, the H(+) initial burst is followed by a slow exponential release of H(+) with a rate constant of 0.017 sec(-1). This rate constant is equal to the steady state rate constant calculated for each heavy meromyosin catalytic site, suggesting that both sites split ATP independently. The magnitude of the H(+) released per ATP bound is always 0.4 in the initial burst, and 0.6 in the slow exponential phase, which equals to a total of 1 H(+) released per ATP hydrolyzed. On the basis of these observations, it is concluded that the H(+) initial burst may well represent a fast ionization of H(+) due to a conformational change as a result of nucleotide binding.