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 conditions favoring the presence of monovanadate, and in the presence of Ca(2+), ADP, and Mg(2+), cleavage results in two fragments of 71- and 38-kDa electrophoretic mobility. On the other hand, under conditions permitting formation of decavanadate, and in the absence of Ca(2+) and ADP, cleavage results in two fragments of 88- and 21-kDa electrophoretic mobility. The amino terminus resulting from cleavage is blocked and resistant to Edman degradation. However, the initial photo-oxidation product can be reduced with NaB(3)H(4,) resulting in incorporation of radioactive (3)H label. Extensive digestion of the labeled protein with trypsin then yields labeled peptides that are specific for the each of the photo-oxidation conditions, and can be sequenced after purification. Collection of the Edman reaction fractional products reveals the radioactive label and demonstrates that Thr(353) is the residue oxidized by monovanadate at the phosphorylation site (i.e. Asp(351)). Correct positioning of monovanadate at the phosphorylation site requires binding of Mg(2+) and ADP to the Ca(2+)-dependent conformation of the enzyme. Subsequent hydrolytic cleavage is likely assisted by the neighboring Asp(601), and yields the 71- and 38-kDa fragments. On the other hand, Ser(186) (and possibly the following three residues: Val(187), Ile(188), and Lys(189)) is the residue that is photo-oxidized by decavanadate in the absence of ADP. Hydrolytic cleavage of the oxidized product at this site is likely assisted by neighboring acidic residues, and yields the 88- and 21-kDa fragments. The bound decavanadate, which we find to produce steric interference with TNP-AMP binding, must therefore extend to the A domain (i.e. small cytosolic loop) in order to oxidize Ser(186). This protein conformation is only obtained in the absence of Ca(2+).
Hua
S
S
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Inesi
G
G
Toyoshima
C
C
D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism* / Q000528:radiation effects; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D010084:Oxidation-Reduction; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D014466:Ultraviolet Rays; D014638:Vanadates / Q000378:metabolism*
D014638:Vanadates; D000244:Adenosine Diphosphate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium