Mechanism of adenosine triphosphate hydrolysis by actomyosin.

The hydrolysis of ATP by acto-HMM has been studied during the transient state using a rapid-mixing apparatus. The rate of substrate binding was slightly slower and the rate of hydrolysis of the first molecule of ATP was essentially the same as for heavy meromyosin (HMM) alone. The rate of acto-HMM dissociation after binding substrate was too fast to measure in a stopped-flow apparatus, consequently dissociation occurs before hydrolysis of the bound ATP. By U nder physiological conditions, namely, 0.1-0.15 M KC1 and greater than 1 mM MgC12, myosin ATPase activity is strongly inhibited. Natural or synthetic actomyosin under these same conditions exists as a precipitate. Addition of ATP usually leads to “clearing,” a decrease in turbidity of the system, which is indicative of dissociation of actin and myosin, at least as measured by physical methods such a flow birefringence or viscosity (Maruyama and Gergely, 1962). The ATPase activity in the clear phase is only slightly higher than that of myosin ATPase. As long as traces of Ca ions are present this phase is followed by precipitation and superprecipitation and a 10to 20-fold increase in ATPase activity. Although this activation is clearly related to the mechanism of contraction, a heterogeneous system is unsuitable for kinetic studies. A better understanding is obtained from studies of the heavy meromyosin-actin complex which remains essentially homogeneous and does not superprecipitate. The fundamental work of Eisenberg and Moos (1968, 1970; Eisenberg et al., 1969) has considerably clarified our understanding of this system. Two competing reactions are involved, the dissociation of actomyosin by ATP and the activation of myosin ATPase by actin. Analysis of the steady-state kinetics by Eisenberg and Moos showed that the behavior could be interpreted as a decrease in the HMM-actin association constant when ATP is bound and a large increase in actin-HMM ATPase relative to myosin. By interpreting the kinetics by a modified MichaelesMenten scheme, the corresponding association and steadystate rate constants were obtained by extrapolating to infinite actin concentration. The real rate of the activated enzyme was found to be about 200 times larger than for myosin. The scheme could be formulated as 20 seo-1 AM + A T P e A . M . A T P A M + A D P + P +AI 1 A 0.1 sec-1 M + A T P e M , A T P A M + ADP + P * From the Department of Biophysics, University of Chicago, Chicago, Illinois 60637. ReceiuedJuly 8, 1971. This work was supported by National Institutes of Health, Grant No. G M 1992, Muscular Dystrophy Association, and Life Insurance Medical Research Fund. E. W. T. acknowledges a Research Career Development award from the U. S . Public Health Service; R. W. L. acknowledges support from the U. S. Public Health Service Training Grant G M 780. t To whom to address correspondence, means of a rapid column separation procedure it was shown that actin combines with the myosin.ADP. P complex and displaces the products of the hydrolysis reaction. It is concluded that this step is responsible for activation of myosin ATPase by actin. A simple kinetic scheme which accounts for the transient and steady-state behavior is presented and compared with the contraction cycle postulated for the sliding filament mechanism. In this scheme hydrolysis occurs largely through the AM * ATP state, Recent studies on the transient state of ATP hydrolysis by myosin and HMM (Lymn and Taylor, 1970) have shown that the first mole of ATP is hydrolyzed at a rapid rate, roughly 100 sec-1, which is much faster than the maximum rate in the Eisenberg and Moos mechanism under comparable conditions. The slow steady-state rate for myosin ATPase was attributed to the rate-limiting dissociation of the products, ADP, and phosphate from the enzyme (Taylor et al., 1970). These studies therefore suggested a modified view of the actin activation mechanism in which actin might affect the rate of product dissociation rather than the hydrolytic step itself. The work presented here describes a study of the HMMactin system in the transient state. A provisional and no doubt oversimplified mechanism is proposed which is consistent with the transient state experiments as well as the steady-state studies of Eisenberg and Moos. The essential feastures of the mechanism are that actomyosin dissociation preceeds hydrolysis, and activation is produced by recombination of actin with the myosin-products complex with displacement of products. The steps in the mechanism can be identified with the contractile cycle postulated in the sliding filament model (Huxley, 1968; Pringle, 1967). Materials and Methods Proteins. Rabbit myosin and heavy meromyosin (HMM)’ were prepared as described previously (Finlayson et al., 1969). Acetone powder was prepared by the method of Tonomura and Yoshimura (1962) and actin was extracted from the powder at 0” by the method of Carsten and Mommaerts (1963). Concentrations of HMM and actin were determined using the difference in absorbance at 291 and 350 mp in 0.5 N NaOH ( e 2 5 1 €360 = 776 cm2/g for HMM; eZg1 €350 = 1150 cm2/g for actin). These figures were obtained by micro-Kjeldahl analyses assuming a nitrogen content of 16.7%:. For the calculations in this paper the molecular weights of HMM and actin are assumed to be 350,000 and 50,000, respectively. ATPase Measurements. The early phase of ATP hydrolysis was measured by determination of [ 32P]phosphate libera1 Abbreviation used is: HMM, heavy meromyosin. B I O C H E M I S T R Y , V O L . 1 0 , N O . 2 5 , 1 9 7 1 4617 L Y M N A N D T A Y L O R column, a three-way stop cock was turned to allow fluid flow from a buffer reservoir driven by a Harvard apparatus pump. The column and buffer were thermostated by circulating water from a temperature bath. Resolution was somewhat poorer than with the technique previously employed of successive layering on top of the resin bed but was adequate for separation of the enzyme-product complex and permitted successive mixing of substrate and actin with the myosin solution.