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Affinity modification of E 1 ‐form of Na + , K + ‐ATPase revealed Asp‐710 in the catalytic site
Author(s) -
Ovchinnikov Yu.A.,
Dzhandzugazyan K.N.,
Lutsenko S.V.,
Mustayev A.A.,
Modyanov N.N.
Publication year - 1987
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(87)81253-x
Subject(s) - chemistry , enzyme , atpase , stereochemistry , atp hydrolysis , protein subunit , hydrolysis , peptide , gamma subunit , covalent bond , biochemistry , organic chemistry , gene
An alkylating ATP analogue, γ‐[4‐( N ‐2‐chlorethyl‐ N ‐methylamino)]benzylamide ATP (ClRATP), covalently binds to the catalytic α‐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 ClRATP was hydrolysed by pepsin under conditions providing maximum stability of the modification product (4°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 α‐subunit polypeptide chain. This fragment located near the γ‐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 E 1 ‐E 2 ATPases. In the Na + ‐(or E 1 ‐)enzyme form Asp‐710 is the target of modification. Evidently E 1 ‐ and E 2 ‐enzymes have different targets in CIRATP modification, i.e. the polypeptide chain regions near the ATP γ‐phosphate in the enzyme active site differ somewhat in their conformations.