The ATP-chase method demonstrated a two-step binding process for ATP to myosin subfragment 1, yielding unambiguous values for K1 and k+2 that are sensitive to experimental conditions.
The initial steps on the myosin ATPase (EC 3.6.1.3) pathway are taken to be: (formula; see text) A two-step binding for ATP is assumed, but the evidence for it is unconvincing; because of the rapidity of the process unambiguous values for K1 and K2 are not available. 2. We investigated the myosin mechanism by the chemical flow-quench technique. Reaction mixtures containing gamma-32PATP plus myosin subfragment 1 were quenched in unlabelled ATP (ATP chase) or acid (Pi burst). 3. We show that the ATP-chase method can lead directly to unambiguous values for K1 and k+2. 4. The binding process was slowed down by 40% ethylene glycol. It was studied as a function of the ATP concentration. A limiting plateau resulted, showing a two-step binding for ATP, and values for K1 and k+2 were obtained. 5. K1 and k+2 are rather sensitive to the experimental conditions. Ethylene glycol and lowering of the pH decrease both constants, but an increase in KCl concentration increases them. This suggests that the binding of ATP to myosin is of an electrostatic nature. 6. The Pi-burst method can lead directly to k+3 + k-3, but under certain conditions the kinetics are governed by K1 and k+2. This uncertainty of the interpretation of Pi-burst experiments is discussed.
Barman et al. (Tue,) conducted a other in Myosin ATPase mechanism. ATP-chase method was evaluated on K1 and k+2 values for ATP binding. The ATP-chase method demonstrated a two-step binding process for ATP to myosin subfragment 1, yielding unambiguous values for K1 and k+2 that are sensitive to experimental conditions.
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