Lithium-7 nuclear magnetic resonance, water proton nuclear magnetic resonance and gadolinium electron paramagnetic resonance studies of the sarcoplasmic reticulum calcium ion transport adenosine triphosphatase

Abstract

The interactions of Gd ion, Li, and 2 substrate analogs, .beta.,.gamma.-imido-ATP (AMP-PNP) and tridentate CrATP, with the Ca2+-ATPase of rabbit muscle sarcoplasmic reticulum were examined by using 7Li+ NMR, water proton NMR, and Gd3+ EPR studies. Steady-state phosphorylation studies indicate that Gd3+ binds to the Ca2+ activator sites on the enzyme with an affinity which is .apprx. 10 times greater than that of Ca2+. 7Li+, which activates the Ca2+-ATPase in place of K+, has been found to be a suitable nucleus for probing the active sites of monovalent cation-requiring enzymes. 7Li+ nuclear relaxation studies demonstrate that the binding of Gd3+ ion to the two Ca2+ sites on Ca2+-ATPase increases the longitudinal relaxation rate (1/T1) of enzyme-bound Li+. The increase in 1/T1 was not observed in the absence of enzyme, indicating that the ATPase enhances the paramagnetic effect of Gd3+ on 1/T1 of 7Li+. Water proton relaxation studies also show that the ATPase binds Gd3+ at 2 tight-binding sites. Titrations of Gd3+ solutions with Ca2+-ATPase indicate that the tighter of the two Gd3+-binding sites (site 1) provides a higher enhancement of water relaxation than the other, weaker Gd3+ site (site 2). The average of the enhancements at the 2 sites is 7.4. These data, together with a titration of the ATPase with Gd3+ ion, yield enhancements, .epsilon.B, of 9.4 at site 1 and 5.4 at site 2. Analysis of the frequency dependence of 1/T1 of water indicates that the electron spin relaxation .tau.s of Gd3+ is unusually long (2 .times. 10-9 s) and suggests that the Ca2+-binding sites on the ATPase experience a reduced accessibility of solvent water. The Ca2+ sites on the Ca2+-ATPase may be buried or occluded within a cleft or channel in the enzyme. The analysis of the frequency dependence is also consistent with 3 exchangeable water protons on Gd3+ at site 1 and 2 fast exchanging water protons at site 2. Addition of the nonhydrolyzing substrate analogs, AMP-PNP and tridentate CrATP, to the enzyme-Gd3+ complex results in a decrease in the observed enhancement, with little change in the dipolar correlation time for Gd3+, consistent with a substrate-induced decrease in the number of fast-exchanging water protons on enzyme-bound Gd3+. From the effect of Gd3+ on 1/T1 of enzyme-bound Li+, Gd3+-Li+ separations of 7.0 and 9.1 .ANG. are calculated. On the assumption of a single Li+ site on the enzyme these distances set an upper limit on the separation between Ca2+ sites on the enzyme of 16.1 .ANG.