Pre-steady-state kinetic evidence for a cyclic interaction of myosin subfragment one with actin during the hydrolysis of adenosine 5'-triphosphate

• Published in: Biochemistry (Easton) Vol. 15; no. 15; pp. 3244 - 3253
• Main Authors: Chock, Stephen P, Chock, P. Boon, Eisenberg, Evan
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 27.07.1976
• Subjects: Actins - metabolism; Adenosine Diphosphate - metabolism; Adenosine Triphosphatases - metabolism; Adenosine Triphosphate - metabolism; Kinetics; Models, Chemical; Myosins - metabolism; Nephelometry and Turbidimetry; Peptide Fragments - metabolism; Phosphates - metabolism; Protein Binding; Scattering, Radiation; Spectrometry, Fluorescence
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00660a013

A single cycle of adenosine 5'-triphosphate (ATP) hydrolysis by a complex of actin and myosin subfragment one (acto-S-1) was studied in a stopped-flow apparatus at low temperature and low ionic strength, using light scattering to monitor the interaction of S-1 with actin and fluorescence to detect the formation of fluorescent intermediates. Our results show that the addition of a stoichiometric concentration of ATP to the acto-S-1 causes a cycle consisting of first, a rapid dissociation of the S-1 from actin by ATP; second, a slower fluorescence change in the S-1 that may be related to the initial phosphate burst; and third, a much slower rate limiting recombination of the S-1 with actin. This latter step equals the acto-S-1 steady-state adenosine 5'-triphosphatase (ATPase) rate at both low and high actin concentrations, and like the steady-state ATPase levels off at a V max of 0.9s-1 at high actin concentration. Therefore, the release of adenosine 5'-diphosphate and inorganic phosphate is not the rate-limiting step in the acto-S-1 ATPase. Rather, a slow first-order step corresponding to the previously postulated transition from the refractory to the nonrefractory state precedes the rebinding of the S-1 to the actin during each cycle of ATP hydrolysis.