Catalysis of an ATP analogue untethered and tethered to lysine 492 of sarcoplasmic reticulum Ca(2+)-ATPase.
2',3'-O-(2,4,6-trinitrophenyl)-8-azido-AMP (TNP-8N3-AMP) and -ATP photolabel Lys-492 at the active site of the Ca(2+)-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-8N3-ATP and the TNP-nucleotide tethered to Lys-492 is stimulated by Ca2+ (kcat = 0.02 s-1, Km = 1.6 microM; k(obs) = 0.08 s-1, respectively, pH 6.0) and exhibits acidic pH optima with shifted pH dependences (pKa = 5.7 and 7.0, respectively). TNP-8N3-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 largely uncoupled from transport; a small, substoichiometric amount of transport is observable at acidic pH. Ca(2+)-dependent phosphorylation of the ATPase with TNP-8N3-ATP is demonstrable under select conditions but with the tethered substrate is too low to be measured with confidence. Neither ADP nor ATP has any effect on the Ca(2+)-dependent catalysis of the tethered nucleotide. Tethering does not appear to affect formation of phosphoenzyme as shown by P(i)-dependent superfluorescence of the tethered nucleotide. The results indicate that Lys-492 is located at the catalytic site within approximately 14 A of Asp-351, which is phosphorylated, and Lys-492 and the adenyl moiety of the nucleotide are closely associated during phosphorylation. Evidently, Lys-492 is not essential for catalysis of phosphoryl transfer, but its movement, specifically separation from the nucleotide, may be critical for coupling with the transport sites.
McIntosh
D B
DB
Department of Chemical Pathology, University of Cape Town Medical School, Observatory, South Africa.
Woolley
D G
DG
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism; D001692:Biological Transport / Q000528:radiation effects; D001894:Borohydrides / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism* / Q000528:radiation effects; D003432:Cross-Linking Reagents; D004795:Enzyme Stability / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008027:Light; D008239:Lysine / Q000378:metabolism; D010766:Phosphorylation; D012519:Sarcoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism* / Q000528:radiation effects
D000345:Affinity Labels; D001894:Borohydrides; D003432:Cross-Linking Reagents; C058476:O(2',3')-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate; D000244:Adenosine Diphosphate; C025364:sodium borohydride; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D008239:Lysine; D002118:Calcium