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 γ-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 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. Copyright (C) 1999 Elsevier Science B.V.