Kinetic studies on hydrolysis of adenosine triphosphate and inosine triphosphate by myosin A

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Abstract

The Michaelis constant (Km) and the maximum velocity (vm) 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 CaCl2, over a wide range of temperature and pH. The following results were obtained:

  • 1.

    1. Both vm and Km 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 vm and Km 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 vm and 7.9 and 2.1 kcal/mole for Km below and above 15°, respectively.

  • 2.

    2. At 20°, the Km of ATPase changed with changing pH in proportion to vm: both Km and vm showed a depression at neutral pH. The pH dependence of the ratio of vm 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 vm and Km were linear and the activation energies of vm and Km were 16.2 and −2.8 kcal/mole, respectively. At pH7, the activation energies of vm and Km, at temperatures higher than 10°, were noticeably different from those at temperatures lower than 10°; these values were for vm, 9.0 and 3.0 kcal/mole, and for Km, −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 (E1S) and an inactive one (E2S), can exist. It was concluded that the complex E2S 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 E1S 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 E1S is independent of pH.

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    To whom inquiries should be sent. Research Institute for Catalysis, Hokkaido University, Sapporo Japan.

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