STRUCTURE FUNCTION ANALYSIS OF THE AMINO-TERMINAL REGION OF THE ALPHA-1 AND ALPHA-2 SUBUNITS OF NA,K-ATPASE/

The alpha 2 isoform of the Na,K-ATPase exhibits kinetic behavior disti nct from that of the alpha 1 isoform. The distinctive behavior is appa rent when the reaction is carried out under conditions (micromolar ATP concentration) in which the K+ deocclusion pathway of the reaction cy cle is rate-limiting; the alpha 1 activity is inhibited by K+, whereas alpha 2 is stimulated. When 32 NH2-terminal amino acid residues are r emoved 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 Blo stein, R. (1994) J. Biol. Chem. 269, 23944-23948). In the current stud y, the region of the alpha 1 NH2 terminus involved in modulating this kinetic behavior has been localized to the highly charged sequence com prising residues 24-32. Within this nonapeptide, differences between a lpha 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), an d (ii) alpha 2 with the first 32 residues identical to the alpha 1 seq uence, alpha 2(1-32 alpha 1), indicates that the distinctive kinetic b ehavior of alpha 1 and alpha 2 is not due to the 24-32 NH2-terminal do main, per se, but rather to its interaction with other, isoform-specif ic region(s) of the alpha 1-protein. We also demonstrate that the dist inct 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 enzym e, and to a higher affinity for ATP. This was determined in studies us ing two approaches: (i) kinetic analysis of the reaction modeled accor ding to a branched pathway of K+ deocclusion through low and high affi nity ATP pathways and, (ii) measurements of the (rapid) phosphorylatio n of the enzyme (E(1) conformation) by [gamma-P-32]ATP following the r ate-limiting formation of the K+-free enzyme from the K+-occluded stat e (E(2)(K) --> E(1) + K+). The observed kinetic differences between al pha 2 and alpha 1 suggest that these Na,K-ATPase isoforms differ in th e steady-state distribution of E(1) and E(2) conformational states.