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

Abstract

An alkylating ATP analogue, y-[4-(N-2-chlorethyl-N-methylamino)]henzylamide ATP (CIRATP), covalently binds to the catalytic a-subunit of Na+,K+-ATPase yielding a product resistant to hydrolysis by the enzyme and inhibiting the ATP-hydrolysing activity. The Naf-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 S). The modified peptide was isolated by HPLC and its amino acid sequence was found to involve residues 706713 of the a-subunit polypeptide chain. This fragment located near the y-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,-E, ATPases. In the Na+-(or E,-)enzyme form Asp-710 is the target of modification. Evidently E,-and E,-enzymes have different targets in ClRATP modification, i.e. the polypeptide chain regions near the ATP y-phosphate in the enzyme active site differ somewhat in their conformations.

Importance of Conserved α-Subunit Segment709GDGVND for Mg2+ Binding, Phosphorylation, and Energy Transduction in Na,K-ATPase

“…Previously, this segment was proposed to engage in ATP binding on basis of chemical labeling with FSBA 1 of Lys 719 (7) or with ␥-[4-(N-2-chlorethyl-N-methylamino)]benzylamide ATP at Asp 710 or Asp 714 (8). Lane et al (9) concluded that Asp 712 and Asp 716 of the rat ␣1-subunit are both essential, because HeLa cells expressing substitutions for Asn did not survive in ouabain medium.…”

Family of human Na+,K+‐ATPase genes Structure of the gene for the catalytic subunit (αIII‐form) and its relationship with structural features of the protein

“…As mentioned above, in the animal EIE2-ATPases this site is evidently formed by conservative regions of the polypeptide chain of the catalytic subunits in the cytoplasmic portion of the enzyme molecules. Analysis of these regions for the presence of permissive consensus sequences [37] and characteristic elements of the secondary structure [38] may evidence that within the nucleotide-binding site of ion-transporting ATPases the B-a" structure (residues 240-265) encoded by the initial part of exon 8 is present in addition to the structure coded by the initial part of exon 16-a target for affinity modification with the ATP analogue [28,[39][40][41]. The data concerning interrelations of intron positions and structural elements of the proposed nucleotide binding domains require thorough analysis and will be published.…”

Low Temperature Molecular Adaptation of the Skeletal Muscle Sarco(endo)plasmic Reticulum Ca2+-ATPase 1 (SERCA 1) in the Wood Frog (Rana sylvatica)

“…Therefore, based on these considerations and the overall sequence identity, we suggest that the nomenclature used for R. sylvatica and R. clamitans Ca 2ϩ -ATPases that are encoded by the cloned cDNAs be SERCA1a as it was proposed earlier for R. esculenta (7). The hydrophobic putative transmembrane domains (M1-M10), the phosphorylation site (residues 349 -355), as well as sites known to bind fluorescein isothiocyanate or the ATP analogues FSBA and CIRATP in SERCA1 (27)(28)(29) are conserved in R. sylvatica SERCA1a. There are five nonconservative amino acid substitutions (Asn, Gln, Ala, Ser, and Leu) characteristic for R. sylvatica (Fig.…”