Chemical modification of an arginine residue in the ATP-binding site of Ca2+ -transporting ATPase of sarcoplasmic reticulum by phenylglyoxal.

Phenylglyoxal (PGO) was used as a reagent for chemical modification of the ATP-binding site of Ca2+ -transporting ATPase of rabbit skeletal muscle sarcoplasmic reticulum (SR-ATPase). When 1 mM PGO was reacted with SR-ATPase at 30 degrees C at pH 8.5, PGO was bound to the ATPase molecule in two-to-one stoichiometry with concomitant loss of activity of the ATPase to form the phosphorylated intermediate (E-P). ATP and ADP prevented the binding of PGO and thereby protected the enzyme from inactivation. The SR membranes were labeled with [14C]PGO and then digested with pepsin to identify the attachment site of PGO. A 14C-labeled peptide (402Ile-Arg*-Ser-Gly-Gln406) was purified to homogeneity by C18-reversed phase HPLC (Arg* denotes the binding site of [14C]PGO). These results indicate that Arg403 is located in the ATP binding site of the SR-ATPase.