Kinetic mechanism of 1-N6-etheno-2-aza-ATP hydrolysis by bovine ventricular myosin subfragment 1 and actomyosin subfragment 1. The nucleotide binding steps.
The large change in fluorescence emission of 1-N6-etheno-2-aza-ATP (epsilon-aza-ATP) has been used to investigate the kinetic mechanism of etheno-aza nucleotide binding to bovine cardiac myosin subfragment 1 (myosin-S1) and actomyosin subfragment 1 (actomyosin-S1). The time course of nucleotide fluorescence enhancement observed during epsilon-aza-ATP hydrolysis is qualitatively similar to the time course of tryptophan fluorescence enhancement observed during ATP hydrolysis. In single turnover experiments, the nucleotide fluorescence rapidly increases to a maximum level, then decreases with a rate constant of 0.045 s-1 to a final level, which is about 30% of the maximal enhancement; a similar fluorescence enhancement is obtained by adding epsilon-aza-ADP to cardiac myosin-S1 or actomyosin-S1 under the same conditions (100 mM KCl, 10 mM 4-morpholinepropanesulfonic acid, 5 mM MgCl2, 0.1 mM dithiothreitol, pH 7.0, 15 degrees C). The kinetic data are consistent with a mechanism in which there are two sequential (acto)myosin-S1 nucleotide complexes with enhanced nucleotide fluorescence following epsilon-aza-ATP binding. The apparent second order rate constants of epsilon-aza-ATP binding to cardiac myosin subfragment 1 and actomyosin subfragment 1 are 2-12 times slower than those for ATP. Actin increases the rate of epsilon-aza-ADP dissociation from bovine cardiac myosin-S1 from 1.9 to 110 s-1 at 15 degrees C which can be compared to 0.3 and 65 s-1 for ADP dissociation under similar conditions. Although there are quantitative differences between the rate and equilibrium constants of epsilon-aza- and adenosine nucleotides to cardiac actomyosin-S1 and myosin-S1, the basic features of the nucleotide binding steps of the mechanism are unchanged.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004985:Ethenoadenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D008433:Mathematics; D009206:Myocardium / Q000201:enzymology*; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D004985:Ethenoadenosine Triphosphate; D015879:Myosin Subfragments; D010446:Peptide Fragments; C012758:aza-epsilon-ATP; D000255:Adenosine Triphosphate; D009218:Myosins