Affinity modification of E1‐form of Na+, K+ ‐ATPase revealed Asp‐710 in the catalytic site

FEBS Letters

Modyanov, N.N.¹  Ovchinnikov, Yu.A.¹  Dzhandzugazyan, K.N.¹  Lutsenko, S.V.¹  Mustayev, A.A.¹ 

[1] Shemyakin Institute of Bioorganic Chemistry, Academy of Science of the USSR, Moscow USSR

关键词: Na+;    K+ -ATPase;    E1-conformation;    Affinity modification;    Catalytic site structure;   

DOI  :  10.1016/0014-5793(87)81253-X

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown