Inhibition of myosin ATPase by metal fluoride complexes

Abstract Magnesium (Mg 2+ ) is the physiological divalent cation stabilizing nucleotide or nucleotide analog in the active site of myosin subfragment 1 (S1). In the presence of fluoride, Mg 2+ 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 γ-phosphate analogs at 1.2 M −1 s −1 with 1 mM MgCl 2 . The observed molar ratio of Mg/S1 in this complex of 1.58 suggests that magnesium occupies the γ-phosphate position in the ATP binding site of S1 (S1–MgADP–MgF x ). The stability of S1–MgADP–MgF x at 4°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 β-phosphate more loosely than the other known phosphate analogs. The structure of S1 in S1–MgADP–MgF x was studied with near ultraviolet circular dichroism, total tryptophan fluorescence, and tryptophan residue 510 quenching measurements. These data suggest that S1–MgADP–MgF x resembles the M**·ADP·P i steady-state intermediate of myosin ATPase. Gallium fluoride was found to compete with MgF x for the γ-phosphate site in S1–MgADP–MgF x . The ionic radius and coordination geometry of magnesium, gallium and other known γ-phosphate analogs were compared and identified as important in determining which myosin ATPase intermediate the analog mimics.