Steady state kinetics at high enzyme concentration. The myosin MgATPase

The rate of ATP hydrolysis by myosin at high concentrations with an ATP-regenerating system increases linearly with increasing added ATP up to a sharp break at the equivalence point of 1 ATP/myosin active site. Theoretical modeling indicates that the data require a KM on the order of the 30 nM value predicted by the rapid kinetic work (Lymn, R. W., and Taylor, E. W. (1970) Biochemistry 7, 2975-2983). Changes in the experimental conditions are found to change the slope of the initial increase in ATPase rate, but not to change the equivalence point. Proteolytic subfragments of myosin do not exhibit a linear initial increase in rate indicating that they are not homogeneous. Purified myosin is also found to show a small additional increase in ATPase rate at much higher ATP levels with a corresponding increase in flux through a pathway with a low extent of oxygen exchange. This high Km component with low oxygen exchange is distinct from the contaminating ATPase reported previously (Sleep, J. A., Hackney, D. D., and Boyer, P. D. (1980) J. Biol. Chem. 255, 4094-4099) which is shown here to be the CaATPase of the sarcoplasmic reticulum.