The active site structure of Na+/K+-transporting ATPase: location of the 5'-(p-fluorosulfonyl)benzoyladenosine binding site and soluble peptides released by trypsin.
When the dog kidney Na+/K+-transporting ATPase (EC 3.6.1.37, formerly EC 3.6.1.3) was labeled with an ATP analogue, 5'-(p-fluorosulfonyl)benzoyladenosine (FSBA), there was a concomitant loss of ATPase activity. The presence of ATP protected the enzyme from both labeling and inactivation. The ATP-sensitive incorporation of FSBA is associated only with modification of the alpha subunit from which two labeled tryptic peptides were purified and sequenced. To establish any regions of the enzyme protruding from the membrane, the native Na+/K+-transporting ATPase from the electric ray, Torpedo californica, was treated with trypsin; and four peptides, which were released into the water phase, were purified and sequenced. A comparison of the peptide sequences with the deduced amino acid sequences of the DNA coding for the alpha subunit of T. californica and sheep kidney reveal the following. (i) FSBA-labeled peptides from the dog kidney enzyme are located in the central hydrophilic domain and show almost complete sequence homology with the same region in the alpha subunit from the electric ray and sheep kidney. Furthermore, the sequence homology of one of the two labeled peptides can be extended to the sarcoplasmic Ca2+-transporting ATPase and B subunit of Escherichia coli K+-transporting ATPase. (ii) Three trypsin-exposed peptides are found in the central hydrophilic domain, and one peptide is in the hydrophilic segment near the C terminus of the alpha subunit. (iii) The active center of Na+/K+-transporting ATPase is likely to be constructed from at least four different stretches in the primary sequence and, irrespective of the different specificity of cations, the various cation transport ATPases that form phosphorylated enzyme appear to have a common structure at the catalytic site for ATP hydrolysis.
D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004285:Dogs; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D008274:Magnesium / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D012995:Solubility; D013861:Thiocyanates; D014101:Torpedo; D014357:Trypsin / Q000378:metabolism
D005452:Fluoresceins; D010446:Peptide Fragments; D013861:Thiocyanates; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D012964:Sodium; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate; D008274:Magnesium; D000241:Adenosine; D011188:Potassium