Binding and hydrolysis of ATP by cardiac myosin subfragment 1: effect of solution parameters on transient kinetics

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Abstract
Transient kinetic data of ATP binding and cleavage by cardiac myosin subfragment 1 (S1) were obtained by fluorescence stopped flow and analyzed by using computer modeling based on a consecutive, reversible two-step mechanism: .**GRAPHIC**. where M1 and M12 denote myosin species with enhanced fluorescence and K''0 K0/(K0[ATP] + 1). The kinetic constants K0, k12, k23, and k32 and the fractional contributions of M1 and M12 to the total fluorescence are analyzed over a range of systematically varied solution parameters. The initial ATP binding equilibrium (Ko), which decreases with increasing pH, is facilitated by a positively charged protein residue with a pK of 7.1. An active-site charge of +1.5 is determined from the ionic strength dependence. The rate constants k12, k23, and k32 also exhibit pK''s near neutrality but increase with increasing pH. The majority of the large (-54 kJ/mol) negative free energy of ATP binding occurs upon S1 isomerization, k12, and a large increase in entropy (183 J/kmol at 15.degree. C) is associated with the cleavage step. The equilibrium constant for the cleavage step, K2, is determined as 3.5 at pH 7.0, 15.degree. C, and 200 mM ionic strength. There are no significant changes in fractional contributions to total fluorescence enhancement due to solvent-dependent conformational changes of S1 in these data. When values for the combined rate constants are calculated and compared with those determined by graphical analysis, it is observed that graphical analysis overestimates the binding rate constant (k0k12) by 25% and the hydrolysis rate constant (k23 + k32) by as much as 30%. Comparison of these data for cardiac S1 with available data for skeletal S1 indicates that the proteins have similar combined rate constants except for k0k12, which is approximately 7 times larger for skeletal S1. As the two proteins'' electrostatic dependencies are similar, a steric constraint to nucleotide binding in the cardiac S1 relative to the skeletal S1 is postulated.