Inhibition of myosin ATPase by metal fluoride complexes

Magnesium (Mg2+) is the physiological divalent cation stabilizing nucleotide or nucleotide analog in the active site of myosin subfragment 1 (S1). In the presence of fluoride, Mg2+ and MgADP form a complex that traps the active site of S1 and inhibits myosin ATPase. The ATPase inactivation rate of the magnesium trapped S1 is comparable but smaller than the other known gamma-phosphate analogs at 1.2 M-1 s-1 with 1 mM MgCl2. The observed molar ratio of Mg/S1 in this complex of 1.58 suggests that magnesium occupies the gamma-phosphate position in the ATP binding site of S1 (S1-MgADP-MgFx). The stability of S1-MgADP-MgFx at 4 degrees C was studied by EDTA chase experiments but decomposition was not observed. However, removal of excess fluoride causes full recovery of the K+-EDTA ATPase activity indicating that free fluoride is necessary for maintaining a stable trap and suggesting that the magnesium fluoride complex is bonded to the bridging oxygen of beta-phosphate more loosely than the other known phosphate analogs. The structure of S1 in S1-MgADP-MgFx was studied with near ultraviolet circular dichroism, total tryptophan fluorescence, and tryptophan residue 510 quenching measurements. These data suggest that S1-MgADP-MgFx resembles the M**.ADP.Pi steady-state intermediate of myosin ATPase. Gallium fluoride was found to compete with MgFx for the gamma-phosphate site in S1-MgADP-MgFx. The ionic radius and coordination geometry of magnesium, gallium and other known gamma-phosphate analogs were compared and identified as important in determining which myosin ATPase intermediate the analog mimics.