Affinity modification of E1‐form of Na+, K+ ‐ATPase revealed Asp‐710 in the catalytic site
- 8 June 1987
- journal article
- research article
- Published by Wiley in FEBS Letters
- Vol. 217 (1) , 111-116
- https://doi.org/10.1016/0014-5793(87)81253-x
Abstract
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 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 CIRATP modification, i.e. the polypeptide chain regions near the ATP γ-phosphate in the enzyme active site differ somewhat in their conformations.Keywords
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