title: Kinetic trapping of intermediates of the scallop heavy meromyosin adenosine triphosphatase reaction revealed by formycin nucleotides.
abstract: The kinetics of interaction of formycin nucleotides with scallop myosin subfragments were investigated by exploiting the fluorescence signal of the ligand. Formycin triphosphate gives a 5-fold enhancement of the emission intensity on binding to heavy meromyosin, and the profile indicates that the kinetics of binding are Ca2+-insensitive. In contrast, the subsequent product-release steps show a marked degree of regulation by Ca2+. In the absence of Ca2+ formycin triphosphate turnover by the unregulated and the regulated heavy meromyosin fractions are clearly resolved, the latter showing a fluorescence decay rate of 0.002 s-1, corresponding to the Pi-release step. In the presence of Ca2+ this step is activated 50-fold. Formycin diphosphate release is also regulated by Ca2+, being activated from 0.008 s-1 to 5 s-1. In contrast with protein tryptophan fluorescence [Jackson & Bagshaw (1988) Biochem. J. 251, 515-526], formycin fluorescence is sensitive to conformational changes that occur subsequent to the binding step and demonstrate, directly, an effect of Ca2+ on both forward and reverse rate constants. Apart from a decrease in the apparent second-order association rate constants, formycin derivatives appear to mimic adenosine nucleotides closely in their interaction with scallop heavy meromyosin and provide a spectroscopic handle on steps that are optically silent with respect to protein fluorescence. A novel mechanism is discussed in which regulation of the formycin triphosphate activity by Ca2+ involves kinetic trapping of product complexes.
authors.affiliation: Department of Biochemistry, University of Leicester, U.K.
authors.forename: A P
authors.initials: AP
authors.lastname: Jackson
authors.forename: C R
authors.initials: CR
authors.lastname: Bagshaw
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D005573:Formycins / Q000378:metabolism*; D007700:Kinetics; D008974:Mollusca / Q000201:enzymology*; D015879:Myosin Subfragments / Q000378:metabolism*; D012265:Ribonucleotides / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence
chemical_list: D000903:Antibiotics, Antineoplastic; D005573:Formycins; D015879:Myosin Subfragments; D012265:Ribonucleotides; C056650:formycin A 5'-(betagamma-imido)triphosphate; C032552:formycin triphosphate; C020332:formycin diphosphate; D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D002118:Calcium