Steady state kinetics at high enzyme concentration. The myosin MgATPase.

D D Hackney,P K Clark

doi:10.1016/s0021-9258(18)89051-2

Abstract

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.

Highlights

The rate of ATP hydrolysis by myosin at high con- and illustratesthe influence of various modifiers of enzymatic centrations with anATP-regeneratingsystem in- activity on thekinetics in this region
The ATPase rate increases linearly at low ATP until a sharp break occurs at the equivalence point of 1ATP/myosin head group as determined from the protein concentration and molecular weighotf myosin
The initial slope is modifiedby changes in pH and ionic strength, but the initial rise remains linear and still breaks sharply a t the same equivalencepoint. This isobserved even under conditions which result in aggregation of the myosin into filaments (30 mhA KCl, pH 8),indicating that no asymmetryis introduced by this aggregation.The same equivalence point is seen in the free ATP level at pH 7 and high salt

Summary

THE MYOSIN MgATPase*

From the Department of ~iologicalSciences, Curneg ~ - ~ e ~ lonUniuers ~ ~ , F iP~ tes ~& ~ r~g hu, a15n21i3a. The rate of ATP hydrolysis by myosin at high con- and illustratesthe influence of various modifiers of enzymatic centrations with anATP-regeneratingsystem in- activity on thekinetics in this region. Creases linearly with increasing added ATP up to a Rapid kinetic work from a number of laboratories Sharp break at the equivalence point of 1 ATPImyosi9n-12) has established a detailed scheme for hydrolysis of ATP active site. For the purpose of this treatment, the data require a XM on the order of the 30 nM value mechanism of Equation 1will suffice.

ATP kl

Pase rate is determineads a function of the amount of added

Myosin ATPase

Findings

Starting Panolpyrwate