CATALYSIS OF AN ATP ANALOG UNTETHERED AND TETHERED TO LYSINE-492 OF SARCOPLASMIC-RETICULUM CA2-ATPASE()

2',3'-O-(2,4,6-trinitrophenyl)-8-azido-A (TNP-8N(3)- AMP) and -ATP pho tolabel Lys-492 at the active site of the Ca2+ ATPase of sarcoplasmic reticulum (McIntosh, D. B., Woolley, D. G., and Berman, M. C. (1992) J . Biol, Chem. 267, 5301-5309). We now find that the hydrolysis of the gamma-phosphate of both TNP-8N(3)-ATP and the TNP-nucleotide tethered to Lys-492 is stimulated by Ca2+ (k(cat) = 0.02 s(-1), K-m = 1.6 mu M; k(obs) = 0.08 s(-1), respectively, pH 6.0) and exhibits acidic pH opt ima with shifted pH dependences (pk(a) = 5.7 and 7.0, respectively). T NP-8N(3)-ATP supports Ca2+ transport with a coupling stoichiometry of 2:1 in the pH range 5.0-7.5. Hydrolysis of the tethered substrate is l argely uncoupled from transport; a small, substoichiometric amount of transport is observable at acidic pH. Ca2+-dependent phosphorylation o f the ATPase with TNP-8N(3)-ATP is demonstrable under select condition s but with the tethered substrate is too low to be measured with confi dence. Neither ADP nor ATP has any effect on the Ca2+ dependent cataly sis of the tethered nucleotide. Tethering does not appear to affect fo rmation of phosphoenzyme as shown by P-i-dependent superfluorescence o f the tethered nucleotide. The results indicate that Lys-492 is locate d at the catalytic site within approximately 14 Angstrom of Asp-351, w hich is phosphorylated, and Lys-492 and the adenyl moiety of the nucle otide are closely associated during phosphorylation. Evidently, Lys-49 2 is not essential for catalysis of phosphoryl transfer, but its movem ent, specifically separation from the nucleotide, may be critical for coupling with the transport sites.