The characterization of myosin-product complexes and of product-release steps during the magnesium ion-dependent adenosine triphosphatase reaction.
Evidence is presented that the myosin subfragment-1-ADP complex, generated by the addition of Mg(2+) and ADP to subfragment 1, is an intermediate within the myosin Mg(2+)-dependent adenosine triphosphatase (ATPase) turnover cycle. The existence of this species as a steady-state intermediate at pH8 and 5 degrees C is demonstrated by fluorescence measurements, but its concentration becomes too low to measure at 21 degrees C. This arises because there is a marked temperature-dependence on the rate of the process controlling ADP dissociation from subfragment 1 (rate=1.4s(-1) at 21 degrees C, 0.07s(-1) at 5 degrees C). In the ATPase pathway this reaction is in series with a relatively temperature-insensitive process, namely an isomerization of the subfragment-1-product complex (rate=0.055s(-1) at 21 degrees C, 0.036s(-1) at 5 degrees C). By means of studies on the P(i) inhibition of nucleotide-association rates, a myosin subfragment-1-P(i) complex was characterized with a dissociation equilibrium constant of 1.5mm. P(i) appears to bind more weakly to the myosin subfragment-1-ADP complex. The studies indicate that P(i) dissociates from subfragment 1 at a rate greater than 40s(-1), and substantiates the existence of a myosin-product isomerization before product release in the elementary processes of the Mg(2+)-dependent ATPase. In this ATPase mechanism Mg(2+) associates as a complex with ATP and is released as a complex with ADP. In 0.1m-KCl at pH8 1.0mol of H(+) is released/mol of subfragment 1 concomitant with the myosin-product isomerization or P(i) dissociation, and 0.23 mol of H(+) is released/mol of subfragment when ATP binds to the protein, but 0.23 mol of H(+) is taken up again from the medium when ADP dissociates. Within experimental sensitivity no H(+) is released into the medium in the step involving ATP cleavage.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000249:Adenosine Monophosphate; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002845:Chromatography; D004492:Edetic Acid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008954:Models, Biological; D009218:Myosins / Q000378:metabolism*; D010710:Phosphates; D011485:Protein Binding; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors
D010710:Phosphates; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D000251:Adenosine Triphosphatases; D009218:Myosins; D008274:Magnesium