CHARACTERIZATION OF STABLE BERYLLIUM FLUORIDE, ALUMINUM FLUORIDE, AND VANADATE CONTAINING MYOSIN SUBFRAGMENT-1 NUCLEOTIDE COMPLEXES

Beryllium and aluminum fluorides are good phosphate analogues. These compounds, like orthovanadate, form stable complexes with myosin subfragment 1 (S1) in the presence of MgADP. The formation of the stable S1-nucleotide complexes is characterized by the loss of ATPase activity. For the complete loss of ATPase activity there was necessary a higher concentration of aluminum than of beryllium or vanadate. In the presence of MgATP the onset of the inhibition is delayed, which indicates that stable complexes cannot form when a specific site is occupied by the gamma-phosphate of ATP or by P(i) derived from the gamma-phosphate. The half-lives of the S1-MgADP-(BeF3-), S1-MgADP-(AlF4-), and S1-MgADP-Vi complexes at 0-degrees-C are 7, 2, and 4 days, respectively. In the presence of actin the rate of decomposition of all of the complexes is significantly enhanced; however, the order of decomposition is reversed, the fastest rate being observed with beryllium and the slowest with aluminum. The formation of the S1-MgADP-(BeF3-) and S1-MgADP-(AlF4-) Complexes is accompanied by an increase in tryptophan fluorescence similar to that observed upon addition of MgATP to S1. The fluorescence increase develops rather slowly, by suggesting that the rate-limiting step in the formation of the stable complex is an isomerization. The rate of the fluorescence change accompanying the formation of the Be complex is faster than that for the Al complex. Addition of vanadate to S1 causes a static quenching of the tryptophan fluorescence. When ADP was added to S1-vanadate, there was a fast transient fluorescence increase followed by a slow decrease. The transient increase probably accompanies the appearance of the S I-MgADP complex, while the decrease is indicative of the formation of the stable S1-MgADP-Vi complex. The activation energy for the formation of the complexes, which was estimated from the temperature dependence of the fluorescence change, is about twice as high for the Al complex as for the other two stable complexes. The rate of formation of the Be and vanadate complexes displays a hyperbolic dependence on Be2+ and vanadate concentration, respectively, while the concentration dependence of the formation of the Al complex is rather complicated and has at least two components. The Be- and Al-S1 complexes, as opposed to uncomplexed S1, are not cleaved by vanadate-induced photocleavage at sites which are located at or in the proximity of the "consensus" ATP-binding site of myosin, viz., at 23 and 31 kDa from the N terminus. The results indicate that the three stable complexes are good analogues of the M**-MgADP-P(i) transition state of the myosin-catalyzed ATP hydrolysis, and their study can help to elucidate the mechanism of the process.