Transient-kinetic studies of the adenosine triphosphatase activity of scallop heavy meromyosin.
Fluorescence stopped-flow experiments were performed to elucidate the elementary steps of the ATPase mechanism of scallop heavy meromyosin in the presence and in the absence of Ca2+. ATP binding and hydrolysis, as monitored by the change in tryptophan fluorescence, appear to be Ca2+-insensitive, whereas both Pi release and ADP release are markedly suppressed in the absence of Ca2+. Rate constants for Pi release are 0.2 s-1 and 0.002 s-1 and for ADP release are 6 s-1 and 0.01 s-1 in the presence and in the absence of Ca2+ respectively. Ca2+ binding to the specific site of the regulatory domain is rapid and its release occurs at 25 s-1, consistent with the time scale of a twitch of the striated adductor muscle. Nucleotide binding is a multi-step process requiring a minimum of three states. In such a model Ca2+ controls the rate of conformational changes at the active site in both the forward and the reverse direction, leading to a large dependence of the rate of nucleotide release, but a lesser effect on the overall equilibrium position. The kinetic trapping of nucleotides and Pi at the active site, in the absence of Ca2+, appears to be a fundamental step in suppressing the interaction of the myosin head with the thin filaments in relaxed molluscan muscle.
Jackson
A P
AP
Department of Biochemistry, University of Leicester, U.K.
Bagshaw
C R
CR
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D005573:Formycins / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D008974:Mollusca / Q000201:enzymology*; D015879:Myosin Subfragments / Q000378:metabolism*; D012265:Ribonucleotides / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D005573:Formycins; D015879:Myosin Subfragments; D012265:Ribonucleotides; C032552:formycin triphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D002118:Calcium