title: Affinity modification of E1-form of Na+, K+-ATPase revealed Asp-710 in the catalytic site.
abstract: An alkylating ATP analogue, gamma-[4-(N-2-chlorethyl-N-methylamino)]benzylamide ATP (C1RATP), covalently binds to the catalytic alpha-subunit of Na+, K+-ATPase yielding a product resistant to hydrolysis by the enzyme and inhibiting the ATP-hydrolysing activity. The Na+-form of the membrane-bound Na+, K+-ATPase modified with C1RATP was hydrolysed by pepsin under conditions providing maximum stability of the modification product (4 degrees C, pH 1.5). The modified peptide was isolated by HPLC and its amino acid sequence was found to involve residues 706-713 of the alpha-subunit polypeptide chain. This fragment located near the gamma-phosphate of ATP is a component of the active site. It is highly homologous with corresponding regions of the catalytic subunits of all the known E1-E2 ATPases. In the Na+-(or E1-)enzyme form Asp-710 is the target of modification. Evidently E1- and E2-enzymes have different targets in C1RATP modification, i.e. the polypeptide chain regions near the ATP gamma-phosphate in the enzyme active site differ somewhat in their conformations.
mesh_terms: D000251:Adenosine Triphosphatases / Q000032:analysis; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000345:Affinity Labels / Q000378:metabolism / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D007668:Kidney / Q000201:enzymology; D009928:Organ Specificity; D010455:Peptides / Q000032:analysis; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013045:Species Specificity; D013552:Swine
chemical_list: D000345:Affinity Labels; D010455:Peptides; D001224:Aspartic Acid; C025165:ATP-gamma-4-(N-2-chloroethyl-N-methylamino)benzylamidate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000254:Sodium-Potassium-Exchanging ATPase