Kinetic trapping of intermediates of the scallop heavy meromyosin adenosine triphosphatase reaction revealed by formycin nucleotides.
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.
Jackson
A P
AP
Department of Biochemistry, University of Leicester, U.K.
Bagshaw
C R
CR
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
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