The alpha 2 isoform of the Na,K-ATPase exhibits kinetic behavior distinct from that of the alpha 1 isoform. The distinctive behavior is apparent when the reaction is carried out under conditions (micromolar ATP concentration) in which the K+ deocclusion pathway of the reaction cycle is rate-limiting; the alpha 1 activity is inhibited by K+, whereas alpha 2 is stimulated. When 32 NH2-terminal amino acid residues are removed from alpha 1, the kinetic behavior of the mutant enzyme (alpha 1M32) is similar to that of alpha 2 (Daly, S. E., Lane, L. K., and Blostein, R. (1994) J. Biol. Chem. 269, 23944-23948). In the current study, the region of the alpha 1 NH2 terminus involved in modulating this kinetic behavior has been localized to the highly charged sequence comprising residues 24-32. Within this nonapeptide, differences between alpha 1 and alpha 2 are conservative and are confined to residues 25-27. The behavior of two chimeric enzymes: (i) alpha 1 with the first 32 residues identical to the alpha 2 sequence, alpha 1 (1-32 alpha 2), and (ii) alpha 2 with the first 32 residues identical to the alpha 1 sequence, alpha 2(1-32 alpha 1), indicates that the distinctive kinetic behavior of alpha 1 and alpha 2 is not due to the 24-32 NH2-terminal domain, per se, but rather to its interaction with other, isoform-specific region(s) of the alpha 1-protein. We also demonstrate that the distinct K+ activation profiles of either alpha 2 or alpha 1M32, compared to alpha 1 is due to a faster release of K+ from the K+-occluded enzyme, and to a higher affinity for ATP. This was determined in studies using two approaches: (i) kinetic analysis of the reaction modeled according to a branched pathway of K+ deocclusion through low and high affinity ATP pathways and, (ii) measurements of the (rapid) phosphorylation of the enzyme (E(1) conformation) by [gamma-P-32]ATP following the rate-limiting formation of the K+-free enzyme from the K+-occluded state (E(2)(K) --> E(1) + K+). The observed kinetic differences between alpha 2 and alpha 1 suggest that these Na,K-ATPase isoforms differ in the steady-state distribution of E(1) and E(2) conformational states.
