LI-7 NUCLEAR MAGNETIC-RESONANCE, WATER PROTON NUCLEAR MAGNETIC-RESONANCE, AND GADOLINIUM ELECTRON-PARAMAGNETIC RESONANCE STUDIES OF THE SARCOPLASMIC-RETICULUM CALCIUM-ION TRANSPORT ADENOSINE-TRIPHOSPHATASE

The interactions of gadolinium ion, lithium, and two substrate analogues,β,γ-imido-ATP (AMP-PNP) and tridentate CrATP, with the calcium ion transport adenosine triphosphatase (Ca2+-ATPase) of rabbit muscle sarcoplasmic reticulum have been 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—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 two 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) and also indicate that the average of the enhancements at the two sites is 7.4. These data, together with a titration of the ATPase with Gd3+ ion, yield enhancements, εB, 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 rs of Gd3+ is unusually long (2 X 10-9 s) and suggests that the Ca2+-binding sites on the ATPase experience a reduced accessibility of solvent water. This may indicate that the Ca2+ sites on the Ca2+-ATPase are buried or occluded within a cleft or channel in the enzyme. The analysis of the frequency dependence is also consistent with three exchangeable water protons on Gd3+ at site 1 and two fast exchanging water protons at site 2. Addition of the nonhydrolyzing substrate analogues, 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 Å 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 Å. © 1979, American Chemical Society. All rights reserved.