Protein-bound ATP: properties of a key intermediate of the magnesium-dependent subfragment 1 ATPase from rabbit skeletal muscle

Abstract

The time course of formation and decay of protein-bound adenosine 5'-triphosphate (ATP) has been monitored during single turnovers of the myosin subfragment 1 ATPase with nonspectrophotometric techniques. The rate constant controlling the ATP cleavage step increases markedly with ionic strength, so that in low salt the protein--ATP complex is observed transiently at higher concentration than the protein-products complex. The kinetics of the ATP cleavage step in a single turnover of the actosubfragment 1 ATPase indicates that under appropriate conditions this step is partially rate limiting during overall steady-state ATPase activity. It follows that a binary subfragment 1-ATP complex is a significant component of the steady-state intermediate of the actosubfragment 1 ATPase. Transient kinetic studies of ATP and adenosine 5'-(3-thiotriphosphate) [ATP (gamma S)] binding show directly that a substrate-induced protein isomerization accompanies ligand binding. The rate constant of the isomerization is 170 s-1 at pH 7.0, 15 degrees C, and 0.01 M ionic strength. Under these conditions nucleotide binding appears to be accompanied by a protein fluorescence increase that is 50% of the increase associated with magnesium-dependent steady-state ATPase activity.

The dynamics of actin and myosin association and the crossbridge model of muscle contraction

“…At low ionic strength the affinity of actin for Sl nucleotide complexes is greatly increased, but the rate of the hydrolysis step [34], except * from [32] and t from [93] becomes sufficiently slow for S1 ATP to be a major component of the steady state [36][37][38].…”

Evidence for the two-step binding of ATP to myosin subfragment 1 by the rapid-flow-quench method

“…Thus, whereas Bagshaw et al (1974) maintain that they are mainly due to M* ATP, Chock et al (1979) and Taylor (1979) conclude that they are due to M**.ADP-P1. Cieeves & Trentham (1982) suggest that the two complexes fluoresce equally well. This uncertainty makes fluorescence stopped-flow studies inadequate for determining the mode of the fixation of ATP to myosin subfragment 1.…”

“…A way of establishing a two-step binding is to show that the kinetics of the overall process (steps 1 and 2, above) follow a hyperbolic relationship with substrate concentration. Therefore M* .ATP must be estimated, and, as stressed by Chock et al (1979) and Geeves & Trentham (1982), the only way of doing this is by the ATP-chase method, in which reaction mixtures containing myosin plus radioactive ATP of various ages are quenched in a large molar excess of unlabelled ATP. This method depends on k-2 being very small (Mannherz et al, 1974;Trentham et al, 1976;Taylor, 1979).…”

Effect of N-Terminal Extension of Cardiac Troponin I on the Ca2+ Regulation of ATP Binding and ADP Dissociation of Myosin II in Native Cardiac Myofibrils

“…To understand the ATPase cycle of actomyosin, transient kinetics and mechanical force measurements have been studied with actomyosin S1 27–39 , myofibrils 40–49 , and muscle fibers 48, 50, 51 . Extensive studies have been done with actomyosin subfragment-1 (S1) and -heavy meromyosin (HMM).…”