Protein-bound adenosine 5'-triphosphate: properties of a key intermediate of the magnesium-dependent subfragment 1 adenosinetriphosphatase from rabbit skeletal muscle.
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.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase; D007536:Isomerism; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000378:metabolism*; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D015879:Myosin Subfragments; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins