Solvent accessibility of the adenosine 5'-triphosphate catalytic site of sarcoplasmic reticulum calcium ATPase

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Abstract
The CaATPase of rabbit skeletal [muscle] sarcoplasmic reticulum was labeled at or near the ATP catalytic site with fluoresceinyl isothiocyanate (FITC), and the accessibility of the attached probe to the bulk solvent was determined by I- quenching of its fluorescence. The quenching of free FITC was also measured. In both cases, the quenching was of the Stern-Volmer type and collisional quenching rate constants were obtained over the pH range 5-8 in the presence of ethylene glycol bis(.beta.-aminoethyl ether)-N,N,N'',N''-tetraacetic acid and with added Ca2+, vanadate, or phosphate. The fluorescence intensity and susceptibility to quenching by I- of free FITC were insensitive to the added ligands. In all cases, the intensity decreased with pH, as predicted from the known properties of FITC mono- and dianions. The collisional quenching rate constants increased at lower pH, as expected for I- quenching for a molecule with decreasing negative charge due to protopation. When FITC was attached to the CaATPase, the FITC fluorescence intensity and I- collisional quenching rate constants were sensitive to ligand binding as well as pH. The changes in fluorescence intensity with acidity, when compared to the results for free FITC, indicated the pKa of the FITC was reduced 0.6 unit when it was attached to the CaATPase. Excited-state lifetime measurements indicated that ligand effects at constant pH were not due to protonation-induced changes in FITC quantum yield but to conformational changes of CaATPase. The ligand-induced changes in the collisional quenching rate constants appeared to be due to changes in steric hindrance to I- colliding with bound FITC rather than changes in local charge near the probe. At all pH values, the hindrance to I- quenching of the fluorescence of the FITC at the ATP binding site was vanadoenzyme < enzyme < phosphoenzyme. The effect of Ca2+ binding to the CaATPase was to decrease the hindrance. The lower the pH, the greater was the decrease caused by Ca2+ binding. At pH 6.0, the transition from E-P to ECa2 was consistent with an increase of 48% in the accessibility of the ATP binding site to the bulk solvent.

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