KINETIC MECHANISM OF MYOFIBRIL ATPASE

The kinetic mechanism of myofibril ATPase was investigated using psoas and mixed back muscle over a range of ionic strengths. Myofibrils were labeled with pyrene iodoacetamide to measure the rate constants for the binding of ATP and formation of the weakly attached state. The velocity of shortening was measured by stopping the contraction at various times by mixing with pH 4.5 buffer. The transient and steady-state rates of ATP hydrolysis were measured by the quench flow method. The results fitted the kinetic scheme [GRAPHICS] The rate constants (or equilibrium constants for steps 1 and 6) were obtained for the six steps. k(5) was calculated from the K-M for shortening velocity, K-1, and k(2). The rate constants were essentially equal for myofibrils and acto-S-1 at low ionic strength. Increasing the ionic strength up to 100 mM in NaCl increased the rate of the hydrolysis step and the size of the phosphate burst and the effective rate of product release became the rate-limiting step. The step size calculated from the velocity of shortening, k(5), and k(2) is 15 nm, based on a model in which step 4 is the force-generating step.