A kinetic scheme for the early phase of ATP hydrolysis by actomyosin ATPase and its bioenergetic implications.

ABSTRACT

By following the absorption pattern of the dye orthocresol red in stopped flow spectrophotometer we have studied the H+ liberation in the early phase of ATP hydrolysis by myosin and actomyosin ATPases at different molar ratios of ATPATPase. In the case of myosin alone, we observe alkalination up to a molar ratio of 101 and net acidification above 301. In the case of actomyosin, hydrolysis results in acidification for all ratios. Estimation of Pi generated in the early phase, employing a coupled enzyme system, gives Pi early burst magnitude of 6.2/head of myosin. Interpreting alkalination as release of HPO2(4-) unaccompanied by H+ in the case of myosin for molar ratios less than 101 together with the results of Pi estimation we deduce that between 6 to 10, H+ ions are withheld by myosin in the early burst phase. Polymerized actin was found to induce concomitant release of H+ during the early phase of ATP hydrolysis. A kinetic scheme is proposed for actomyosin ATPase which encompasses the pre-steady state as well. Bioenergetic significance of these protons held by the myosin heads for the process of muscular contraction is discussed.