The characterization of myosin–product complexes and of product-release steps during the magnesium ion-dependent adenosine triphosphatase reaction

Abstract

Evidence is presented that the myosin subfragment-1–ADP complex, generated by the addition of Mg2+ and ADP to subfragment 1, is an intermediate within the myosin Mg2+-dependent adenosine triphosphatase (ATPase) turnover cycle. The existence of this species as a steady-state intermediate at pH8 and 5°C is demonstrated by fluorescence measurements, but its concentration becomes too low to measure at 21°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°C, 0.07s-1 at 5°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°C, 0.036s-1 at 5°C). By means of studies on the Pi inhibition of nucleotide-association rates, a myosin subfragment-1–Pi complex was characterized with a dissociation equilibrium constant of 1.5mm. Pi appears to bind more weakly to the myosin subfragment-1–ADP complex. The studies indicate that Pi 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 Mg2+-dependent ATPase. In this ATPase mechanism Mg2+ 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 Pi 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.