Cryoenzymic studies on actomyosin ATPase. Evidence that the degree of saturation of actin with myosin subfragment 1 affects the kinetics of the binding of ATP

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Abstract
The initial steps of actomyosin subfragment 1 (acto-S1) ATPase (dissociation and binding of ATP) were studied at -15.degree. C with 40% ethylene glycol as antifreeze. The dissociation kinetics were followed by light scattering in a stopped-flow apparatus, and the binding of ATP was followed by the ATP chase method in a rapid-flow quench apparatus. The data from the chase experiments were fitted to E + ATP .dblarw. (K1) E .cntdot. ATP .fwdarw. (k2) E*ATP, where E is acto-S1 or S1. The kinetics of the binding of ATP to acto-S1 were sensitive to the degree of saturation of the actin with S1. There was a sharp transition with actin nearly saturated with S1: when the S1 to actin ratio was low, the kinetics were fast (K1 > 300 .mu.M, k2 > 40 s-1); when it was high, they were slow (K1 = 14 .mu.M, k2 = 2 s-1). With S1 alone K1 = 12 .mu.M and k2 = 0.07 s-1. With acto heavy meromyosin (acto-HMM) the binding kinetics were the same as with saturated acto-S1, regardless of the HMM to actin ratio. The dissociation kinetics were independent of the S1 to actin ratio. Saturation kinetics were obtained with Kd = 460 .mu.M and kd = 75 s-1. The data for the saturated acto-S1 could be fitted to a reaction scheme, but for lack of structural information the abrupt dependence of the ATP binding kinetics upon the S1 to actin ratio is difficult to explain. It is tentatively proposed that the transition is due to a cooperative phenomenon involving head-head interaction. It is suggested that in unsaturated acto-S1 the heads do not interact, even when attached to adjacent actin monomers, but that as further S1 binds and the saturation nears completion there is an abrupt structural change of the filament which is propagated along it. This is then transmitted to the heads which now interact. Therefore, it is proposed that when the heads interact, the ATP binding kinetics are slow (apparently this is always the case with acto-HMM) but when they do not, the kinetics are fast. The kinetic transition is not due to the presence of ehtylene glycol: it was also obtained in an aqueous buffer with N6-ethenoadenosine 5''-triphosphate. The results are discussed with reference to the structural information available in the literature.