title: Evidence for the two-step binding of ATP to myosin subfragment 1 by the rapid-flow-quench method.
abstract: 1. 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-32P]ATP 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.
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D019855:Ethylene Glycol; D005026:Ethylene Glycols; D007700:Kinetics; D008954:Models, Biological; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D011189:Potassium Chloride; D011485:Protein Binding
chemical_list: D005026:Ethylene Glycols; D015879:Myosin Subfragments; D010446:Peptide Fragments; D010761:Phosphorus Radioisotopes; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D009218:Myosins; D019855:Ethylene Glycol