Description
Mechanistic exploration of the Na⁺-pump from electrically excitable tissue was carried out on two levels, a NMR and mass spectral kinetic ¹⁸0 analysis of a key step associated with transmembrane K⁺-K⁺ exchange and probes at the nature of histidyl involvement in the catalytic and associated subsites of the enzyme. Microsomal preparations of the (Na⁺,K⁺)-ATPase from Electrophorus electroplax catalyze a rapid Mg⁺⁺- and K⁺- dependent exchange of water oxygens with inorganic phosphate. The phosphate-water oxygen exchange was monitored using the oxygen-18 shift on the ³¹P-NMR signal of inorganic phosphate. It was found that the phosphate exchanges no more than one oxygen atom before dissociating from the enzyme but that, at more acidic pHs, the mode of the exchange is modified to involve more than one oxygen release per binding-dissociation event of phosphate to the enzyme. Mathematical treatment of the data generated theoretical plots of the time course of the exchange process and yielded a rate constant for the exchange reaction. Na⁺, an inhibitor of the exchange, did not modify the basic kinetic pattern of the reaction. Rose bengal-sensitized photooxidation and ethoxyformylation of microsomal preparations of the (Na⁺, K⁺)-ATPase from electroplax and dog kidney outer medulla demonstrated the presence of two spatially distinct histidyl residues in the active site of the enzyme. One of the residues (histidine I), critical for the integrity of the overall ATP hydrolysis and the p-NPPase activities, is probably situated in the high affinity ATP binding site of the enzyme and its reactivity towards the two modification methods is independent of the conformation of the enzyme (E₁ versus E₂ form). Modification of the other histidyl residue (histidine II) is necessary but not sufficient for inactivation of the p-NPPase reaction catalyzed by the enzyme, and does not have any effect on the overall ATP hydrolytic activity; histidine II is probably located in a subsite of the catalytic site that accommodates the artificial substrate p-NPP, and its reactivity is modulated by the conformation of the enzyme (in the E₂-K⁺ conformation of the enzyme, the residue is much less susceptible to modification).
