The regulation of the rate of ATP hydrolysis by H-meromyosin

Abstract

The effect of N-ethylmaleimide on the ATPase activity and ADP binding of tryptic H-meromyosin was studied at 6 and 23 °C temperatures. The affinity constant of H-meromyosin for ADP with Mg as activator was increased by small concentrations of N-ethylmaleimide (2.25 moles per mole of enzyme) at both temperatures, accompanied by activation of ATP hydrolysis at 25 °C and inhibition at 6 °C. With higher N-ethylmaleimide concentrations, the ATPase activity was inhibited at both temperatures, without comparable inhibition of ADP binding. Rapid kinetic analysis of the rate of development of difference spectrum after the addition of ATP or ADP to H-meromyosin indicates, that blocking of the S 1 and S 2 SH groups of H-meromyosin decreases both the formation ( k 1) and the dissociation ( k 2) rate constants of H-meromyosin substrate complex. At 6 °C, in the presence of Mg, the value of k 2 for ADP is similar to the turnover number of ATP hydrolysis, suggesting that dissociation of ADP from the active site may be the rate-limiting step of ATP hydrolysis. At 23 °C, the turnover number of Mg-moderated ATP hydrolysis is much smaller than k 2, indicating that the rate limitation shifted so another, so far unidentified, step.