Reaction of (Na-K)ATPase with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole: evidence for an essential tyrosine at the active site.
The reaction of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole [NBD-Cl] with purified eel electrophax Na+ and K+ stimulated adenosine triphosphatase [(Na-K)ATPase] has been monitored by changes in the (Na-K)ATPase activity, the K+ stimulated p-nitrophenyl phosphatase [PNPase] activity, and the protein ultraviolet absorption spectrum. The NBD-Cl reacts with two tyrosine residues per mol of enzyme (approximately 6-7 nmol/mg of protein), as judged by changes in protein absorption spectra and incorporation of [14C]NBD-Cl. The modified tyrosine groups are located on the Mr = 95 000 polypeptide chain and react at different rates. Only one tyrosine modification is necessary for complete inhibition of (Na-K)ATPase activity, although both must be modified for complete inhibition of PNPase activity. Reversal of these modifications by 2-mercaptoethanol restores 65% of both activities. Na+ increases the rate of tyrosine modification, K+ decreases the rate, and ATP affords the more reactive tyrosine group complete protection. NBD-Cl modification of approximately 6-7 nmol of tyrosine groups/mg of protein results in a large decrease in ATP affinity as judged by equilibrium binding. These results are compared with similar results obtained from NBD-Cl modification of the coupling factors of oxidative phosphorylation and photophosphorylation. A model is presented suggesting an asymmetric arrangement of two 95 000 polypeptide chains with a single tyrosine residue at the ATP site.
D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D004524:Eels; D004557:Electric Organ / Q000201:enzymology; D007700:Kinetics; D008433:Mathematics; D010069:Oxadiazoles / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D011485:Protein Binding; D012964:Sodium / Q000378:metabolism; D013053:Spectrophotometry; D014443:Tyrosine
D010069:Oxadiazoles; D014443:Tyrosine; D012964:Sodium; D000251:Adenosine Triphosphatases; D009327:4-Chloro-7-nitrobenzofurazan; D011188:Potassium