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The Michaelis constant ( K m ) and the maximum velocity ( v m ) of myosin A-ATPase (EC 3.6.1.3) and of myosin A-ITPase were measured in 0.6 M KCl, and in the presence of 7 mM CaCl 2 , over a wide range of temperature and pH. The following results were obtained: 1. 1. Both v m and K m of ITPase were increased proportionally to each other with increasing pH, and their half-saturation values were obtained at pH 6.8. The Arrhenius plots of v m and K m were readily represented by two reactions of different activation energies and the extrapolations of the linear portions intersected in each case at around 15°. The value of activation energies were 25.6 and 11.9 kcal/mole for v m and 7.9 and 2.1 kcal/mole for K m below and above 15°, respectively. 2. 2. At 20°, the K m of ATPase changed with changing pH in proportion to v m : both K m and v m showed a depression at neutral pH. The pH dependence of the ratio of v m of ITPase to that of ATPase was given by a bell-shaped curve having its maximum at pH 7.5. At 0°, the pH-activity curve of ATPase lacked the neutral depression. At pH 6, the Arrhenius plots of v m and K m were linear and the activation energies of v m and K m were 16.2 and −2.8 kcal/mole, respectively. At pH7, the activation energies of v m and K m , at temperatures higher than 10°, were noticeably different from those at temperatures lower than 10°; these values were for v m , 9.0 and 3.0 kcal/mole, and for K m , −1.9 and −10.4 kcal/mole, below and above 10°, respectively. 3. 3. These results were analyzed according to the mechanism proposed by one of the authors where by, in ATPase, two forms of the Michaelis complex, viz. , an active one (E 1 S) and an inactive one (E 2 S), can exist. It was concluded that the complex E 2 S is not formed in ITPase but in ATPase it is dominant at around pH 7.5, and at higher temperatures. It was further concluded that the rate-constant of dissociation of the Michaelis complex E 1 S into the enzyme and the substrate is much smaller than that of the breakdown of the complex into the products; the rate-constant of the formation of E 1 S is independent of pH.

The behaviours of three synthetic ATP analogues, 6-dimethylamino-9-β- d -ribofuranosylpurine 5′-triphosphate (dimethyl-ATP), 9-(4′-hydroxybutyl)-6-aminopurine 4′-triphosphate (BTP) and ribose 5-triphosphate (RTP), on hydrolysis by myosin A were investigated mainly in 0.5 M KCl and 5 m M CaCl 2 (pH7.0, 20°), together with those of ATP, ITP and inorganic tripolyphosphate (TP i ). The following results were obtained. 1. 1. The effect of divalent cations, such as Mg 2+ , Mn 2+ , Ca 2+ , Sr 2+ and Ba 2+ , on the rate of hydrolysis of BTP, TP i subtrates, the relations between the ionic radii ( r ) of various added divalent cations and the rates of hydrolysis were expressed by a bell-shaped curve, which showed a maximum at r = 0.95 A . 2. 2. The Michaelis constants of hydrolysis of ITP, TP i , BTP, RTP, dimethyl-ATP and ATP were 3, 2.5, 2.5, 1.7, 1.4 and 0.2 and m M , respectively, while the ratios of the maximum velocity of hydrolysis of ITP, BTP, dimethyl-ATP, TP i and RTP to that of ATP were, 7, 3, 1.3, 1 13.5 and 1 20 , respectively. 3. 3. The Michaelis constant and the maximum velocity of hydrolysis of TP i showed a remarkable increase with decreasing pH, while the rate of hydrolysis of RTP depended slightly on the pH. The dependence of the rate of hydrolysis of BTP on the pH showed a slight depression at the neutral pH and its Michaelis constant was independent of the pH. 4. 4. p -Chloromercuribenzoate and EDTA inhibited the hydrolysis of TP i , RTP, ITP, dimethyl-ATP and BTP, although they activated ATPase. These results have been interpreted on the basis of the reaction between myosin A and nucleoside triphosphates which has been proposed by one of the present authors 7,24 and is further discussed below.