Mechanism of adenosinetriphosphatase activity of trinitrophenylated myosin subfragment 1.

The rate and extent of conformational changes induced by nucleotides in trinitrophenylated myosin subfragment 1 (S-1) were studied. The absorbance spectrum of the trinitrophenyl moiety attached to the reactive lysyl residue of S-1 changed considerably upon addition of ATP or its analogues. ATP or AMP-PNP induced a larger spectral change than that of ADP, and its value was independent of the presence of Mg2+. The trinitrophenylation of the reactive lysine considerably decreased the enhancement of the tryptophan fluorescence induced by MgATP, while it did not affect fluorescence enhancement induced by MgADP or MgAMP-PNP. The rate of formation of nucleotide-induced absorbance changes in trinitrophenylated (TNP) S-1 was followed in a stopped-flow spectrophotometer. The reaction could be described by a single exponential at every nucleotide concentration. The calculated apparent first-order rate constant, kobsd, increased with rising concentrations of MgATP, ATP, MgADP, and MgAMP-PNP and finally reached a plateau. The absorbance change that appeared upon addition of MgATP to TNP S-1 eventually decayed in two phases until it reached the level of that induced by MgADP. The nucleotide concentration dependence of kobsd deviated from a hyperbolic function in all cases studied. The nucleotide concentration dependence of the signal amplitude was also measured, and it was found to be independent of the presence of Mg2+. The obtained results deviated from a computer-simulated binding curve, which was computed by assuming one identical binding site for each TNP S-1 molecule. The results indicate that an enzyme-substrate complex is the predominant intermediate in the TNP S-1 catalyzed MgATP hydrolysis, instead of an enzyme-product complex, which is the case with normal, unmodified S-1.