THE CONFORMATIONS OF A FUNCTIONAL SPIN-LABELED DERIVATIVE OF GASTRIC H K-ATPASE INVESTIGATED BY EPR SPECTROSCOPY/

A spin-labeled derivative of porcine gastric H/K-ATPase with high ATP hydrolyzing activity (77 mu mol of P-i/(mg . h)) has been prepared. Ov er 65% of initial ATPase activity (115 mu mol of P-i/(mg . h)) was pre served after complete reaction of the enzyme with the lysine reactive nitroxide spin-labeled TEMPO isothiocyanate (TITC). In contrast, rapid and complete loss of ATPase activity occurred after reaction of the e nzyme with the lysine directed fluorescent probe FITC. Conventional EP R spectra of TITC labeled H/K-ATPase reflected mainly the slow rotatio nal diffusion of the enzyme in the membrane. An upper limit enzyme int ramembranous radius of 108 Angstrom was calculated on the basis of rot ational correlation times estimated from saturation transfer (ST) EPR spectral lineshapes. Conventional EPR spectra exhibited two major comp onents corresponding to at least two populations of strongly constrain ed spin-labels. Difference spectroscopy revealed that the proportion o f these two components changed markedly with temperature. Moreover, th e proportion of the components was sensitive to the presence of the ac tivating ionic ligands Mg2+ and ATP, which induce enzyme conformationa l transitions, and to the reversible inhibitor SCH 28080, which binds to the K+ sensitive form of the enzyme. These findings show that EPR s pectroscopy is able to report functionally coupled conformational chan ges of gastric H/K-ATPase and imply that the spin-labels are attached to lysines within functionally important regions of the enzyme.