The mechanism of the skeletal muscle myosin ATPase. I. Identity of the myosin active sites.

S P Chock,E Eisenberg

doi:10.1016/s0021-9258(18)50748-1

Abstract

In the present study, the question of whether the two myosin active sites are identical with respect to ATP binding and hydrolysis was reinvestigated. The stoichiometry of ATP binding to myosin, heavy meromyosin, and subfragment-1 was determined by measuring the fluorescence enhancement caused by the binding of MgATP. The amount of irreversible ATP binding and the magnitude of the initial ATP hydrolysis (initial Pi burst) was determined by measuring [gamma-32P]ATP hydrolysis with and without a cold ATP chase in a three-syringe quenched flow apparatus. The results show that, under a wide variety of experimental conditions: 1) the stoichiometry of ATP binding ranges from 0.8 to 1 mol of ATP/myosin active site for myosin, heavy meromyosin, and subfragment-1, 2) 80 to 100% of this ATP binding is irreversible, 3) 70 to 90% of the irreversibly bound ATP is hydrolyzed in the initial Pi burst, 4) the first order rate constant for the rate-limiting step in ATP hydrolysis by heavy meromyosin is equal to the steady state heavy meromyosin ATPase rate only if the latter is calculated on the basis of two active sites per heavy meromyosin molecule. It is concluded that the two active sites of myosin are identical with respect to ATP binding and hydrolysis.

Highlights

In the present study, the question of whether the two myosin active sites are identical with respect to ATP binding and hydrolysis was reinvestigated
The stoichiometry of ATP binding to myosin, heavy meromyosin, and subfragment-l was determined by measuring the fluorescence enhancement caused by the binding of MgATP
The results show that, under a wide variety of experimental conditions: 1) the stoichiometry of ATP binding ranges from 0.8 to 1 mol of ATP/myosin active site for myosin, heavy meromyosin, and subfragment-1, 2) 80 to 100%

Summary

IDENTITY OF THE MYOSIN ACTIVE SITES*

ATPase (Received for publication, May 30, 1978, and in revised form, October 10, 1978). The question of whether the two myosin active sites are identical with respect to ATP binding and hydrolysis was reinvestigated. Where, using the nomenclature of Bagshaw et al [3], M is myosin, T is ATP, D is ADP, P, is inorganic phosphate, and * and ** qualitatively represent the amount of fluorescence enhancement shown by the various intermediates In this scheme, ATP binds weakly to myosin to form the collision intermediate Me T which undergoes an essentially irreversible conformational change to M*. The results show that, under a wide variety of conditions, both myosin heads bind ATP irreversibly and 75 to 90% of the irreversibly bound ATP is hydrolyzed in the initial P, burst.

AND METHODS

RESULTS

DISCUSSION