Distinct topologies of mono- and decavanadate binding and photo-oxidative cleavage in the sarcoplasmic reticulum ATPase

UV irradiation of the sarcoplasmic reticulum (SR) ATPase in the presence of vanadate cleaves the enzyme at either of two different sites. Under condit ions favoring the presence of monovanadate, and in the presence of Ca2+, AD P, and Mg2+ cleavage results in two fragments of 71- and 38-kDa electrophor etic mobility. On the other hand, under conditions permitting formation of decavanadate, and in the absence of Ca2+ and ADP, cleavage results in two f ragments of 88- and 21-kDa electrophoretic mobility. The amino terminus res ulting from cleavage is blocked and resistant to Edman degradation. However , the initial photo-oxidation product can be reduced with (NaBH4)-H-3, resu lting in incorporation of radioactive H-3 label. Extensive digestion of the labeled protein with trypsin then yields labeled peptides that are specifi c for the each of the photo-oxidation conditions, and can be sequenced afte r purification. Collection of the Edman reaction fractional products reveal s the radioactive label and demonstrates that Thr(353) is the residue oxidi zed by monovanadate at the phosphorylation site (i.e. Asp(351)). Correct po sitioning of monovanadate at the phosphorylation site requires binding of M g2+ and ADP to the Ca2+-dependent conformation of the enzyme. Subsequent hy drolytic cleavage is likely assisted by the neighboring Asp(601), and yield s the 71- and 38-kDa fragments. On the other hand, Ser(186) (and possibly t he following three residues: Val(187), Ile(188) and Lys(189)) is the residu e that is photo-oxidized by de cavanadate in the absence of ADP. Hydrolytic cleavage of the oxidized product at this site is likely assisted by neighb oring acidic residues, and yields the 88- and 21-kDa fragments. The bound d ecavanadate, which we find to produce steric interference with TNP-AMP bind ing, must therefore extend to the A domain (i.e. small cytosolic loop) in o rder to oxidize Ser(186). This protein conformation is only obtained in the absence of Ca2+.