On the Active Site of Myosin A-Adenosine Triphosphatase*

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Abstract
It was already found that the ATPase [EG 3.6.1.3] activity of myosin in the presence of Mg++ is markedly enhanced by trinitrophenylation of myosin. Thereafter, the reaction mechanism of myosin-ATPase has been elucidated, and it was shown that myosin is a double-headed enzyme which hydrolyzes ATP by two different routes: one is simple hydrolysis of the Michaelis complex (ES), and the other is hydrolysis of ATP via phosphoryl myosin (EP). The purpose of the present paper is to report studies of the kinetic mechanism of enhancement of Mg++-ATPase activity in steady state by trinitrophenylation of myosin, on the basis of the reaction mechanism mentioned above. We obtained the following results. 1. In the presence of 1M KC1 and 20 mM MgCl2, the rate of ATPase of trinitrophenyl (TNP)-myosin in steady state was much higher than that of the control myosin, and Pi was continuously liberated from the TNP-myosin-ATP system at a high rate, about equal to that of the initial burst of P1-liberation observed on the control myosin. 2. Both the ATPase activity of actomyosin type and the super-precipitation of actomyosin by ATP were unaffected by trinitrophenylation of myosin. 3. The rate of ATPase of the control myosin in steady state was independent of the size of the initial burst, but the rate of ATPase of TNP-myosin decreased when the size of initial burst was reduced by p-nitrothiophenylation or prolonged incubation of myosin. The rate of ATPase of TNP-myosin reached the level of control myosin, when the size of the initial burst became zero. From these results, it was concluded that, at least in the presence of Mg++ and a high concentration of KC1, trinitrophenylation of myosin has no significant effect on the simple hydrolysis of ES, but accelerates markedly the decomposition of EP.

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