Replacement of several single amino acid side chains exposed to the inside of the ATP-binding pocket induces different extents of affinity change in the high and low affinity ATP-binding sites of rat Na/K-ATPase

To investigate the relationship between the high and the low affinity ATP-binding site, which appears during the Na+/K+-ATPase reaction, four amino acids were mutated, the side chains of which are exposed to inside of the ATP-binding pocket. Six mutants, F475Y, K480A, K480E, K501A, K501E, and R544A, where the numbers correspond to the pig Na+/K+-ATPase alpha-chain, were expressed in HeLa cells. The apparent affinities were determined by high affinity ATP-dependent phosphorylation and by the low affinity activation of Na+/K+-ATPase or low affinity ATP inhibition of K+-para-nitrophenylphosphatase (pNPPase). For the mutants K480A and K501A, little affinity change was detected for either the high affinity or the low affinity effect. In contrast, the other four mutants reduced both apparent affinities. Strikingly, R544A had a 30-fold greater effect on the high affinity ATP site than the low affinity site. For the F475Y mutant, it is likely that there was a greater effect on the low affinity site than the high affinity site, but for both F475Y and K480E the affinity for the low affinity ATP effect was reduced so much that it was not possible to estimate a K-0.5. However, both the affinities for the K480E were reduced to similar to1/20. The turnover number of the Na+/K+-ATPase and the apparent affinity for Na+ and pNPP was reduced slightly or not at all for these mutants, but the turnover number of K+-pNPPase and the apparent affinity for K+ were increased. These and other data suggest the presence of only one ATP-binding site, which can change its conformation to accept ATP with a high and low affinity. The requirement of Arg-544 and possibly Lys-501 is more important in forming a high affinity ATP binding conformation, and Phe-475 and possibly Lys-480 are more important in forming the low affinity ATP binding conformation.