LYS515-LYS492 CROSS-LINKING BY DIDS INTERFERES WITH SUBSTRATE UTILIZATION BY THE SARCOPLASMIC-RETICULUM ATPASE

Sarcoplasmic reticulum (SR) Ca2+ ATPase was derivatized with 4,4'-diis othiocyanatostilbene-2,2'-sulfonic acid (DIDS), and complete enzyme in activation was produced with a molecular stoichiometry of one DIDS per ATPase. It was determined by peptide analysis and sequencing that Lys 492 and Lys515 were the ATPase residues derivatized by DIDS, Lack of e lectrophoretic resolution of the two peptide fragments that result fro m a single tryptic cut at Arg505 demonstrated that the two derivatized residues were cross-linked. Cross-linking of Lys492 and Lys515 by DID S interfered with ATPase utilization of both ATP and p-nitrophenyl pho sphate substrates, whereas derivatization of only Lys515 with fluoresc ein isothiocyanate interfered with ATPase utilization of ATP but not o f p-nitrophenyl phosphate. Cross-linking with DIDS implies a distance of approximately 13 Angstrom between Lys492 and Lys515, which correspo nds to the length of ATP bound in an extended configuration. Therefore , within the groove of the nucleotide binding domain, the ATP substrat e is positioned with the adenosine moiety near Lys515 and its terminal phosphate near Lys492.