Interaction of Heavy Meromyosin with Substrate

Abstract

Abstract Difference spectra induced by ATP and its analogues of the ultraviolet absorption of heavy meromyosin around 280 mµ were measured by the double cell method. The difference spectrum induced by ATP showed two peaks, at 281 and 289 mµ, and a shoulder near 300 mµ. The maximum value of the difference molar extinction coefficient, ΔE, at 289 mµ, was 5000 ± 300 m-1 cm-1. The difference spectrum caused by ATP near 290 mµ decreased with time after its addition and reached the same value as that induced by ADP. The difference spectrum attributable to ADP showed two peaks at 280 and 288 mµ, but the shoulder near 300 mµ was scarcely observable. The maximum value of ΔE at 288 mµ was 2700 ± 600 m-1 cm-1. AMP, adenosine, adenine, and d-ribose induced slight but detectable difference spectra of heavy meromyosin. Pyrophosphate and sodium triphosphate induced difference spectra similar in shape to that of ADP, but the values of ΔE at 287 mµ were only about 1500 m-1 cm-1. The difference optical density at 288 mµ was measured as a function of ADP concentration in the presence of 0.06 m KCl and 8.3 mm MgCl2. The unit weight of heavy meromyosin was calculated to be 3.65 x 105, after determining the minimum ADP concentration needed to give a maximum spectral change. The decay of change in difference optical density, ΔO.D., induced by ATP was measured as a function of time at a fixed wave length. The initial velocity of steady state ATPase of heavy meromyosin was measured by the pH-stat method, and the rate constant of the step from the enzyme-substrate complex to the products, k2, was evaluated. The decay of ΔO.D. was found to agree well with that expected for the enzyme-substrate complex over a wide range of concentrations of KCl, NaCl, and MgCl2.