Transient kinetic analysis of turnover-dependent fluorescence of 2',3'-O-(2,4,6-trinitrophenyl)-ATP bound to Ca2+-ATPase of sarcoplasmic reticulum.

The fluorescence of 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) bound to the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum is greatly enhanced during turnover induced by ATP plus Ca2+ (Watanabe, T., and Inesi, G. (1982) J. Biol. Chem. 257, 11510-11516). We have studied the kinetics of induction of TNP-ATP fluorescence and of its decay and have found a close correlation with levels of phosphorylated intermediate of the enzyme, E-P. Steady-state kinetic studies suggested competitive binding of ATP and TNP-ATP to the catalytic site, with Km and Ki values of 2.4 and 1.0 microM, respectively. Rate constants for fluorescence enhancement and for E-P formation in the presteady state were 1.2 s-1 or 97-130 s-1 under conditions resulting in TNP-ATP or ATP saturation respectively, of the enzyme at inception of reaction. The slow process was concluded to be the koff for dissociation of TNP-ATP from the catalytic site. Following this dissociation, a second TNP-ATP site was detected, which both formed (97-130 s-1) and decayed (0.22 s-1) synchronously with E-P. TNP-ATP binding to this noncatalytic site was rapid (5 X 10(7) M-1 s-1) and resulted in high fluorescence during steady-state turnover. Fluorescence was found to be dissociated from E-P by KCl (100 mM). KCl had little effect on E-P levels, but decreased fluorescence by 68%. These studies provide independent kinetic evidence for the existence of both catalytic and noncatalytic, or "regulatory," nucleotide-binding sites, but cannot distinguish whether the two sites exist independently or whether the catalytic site is transformed into a regulatory site on phosphorylation. The latter site, which shows relatively high selectivity for TNP-ATP over ATP, and which is simultaneously hydrophobic and freely accessible to the medium, may play a role during energy transduction. The changes occurring at this site during catalysis are conveniently monitored with TNP-ATP fluorescence.