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

Satomi Teramachi,Toshiaki Imagawa,Shunji Kaya,Kazuya Taniguchi

doi:10.1074/jbc.m204772200

Abstract

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 approximately 1/20. 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.

Highlights

Naϩ/Kϩ-ATPase is an integral membrane protein consisting of ␣ and ␤ subunits and couples ATP hydrolysis to the active transport three Naϩ and two Kϩ ions against their electrochemical gradients
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 K0.5
The data show that each single amino acid substitution, such as F475Y, K480E, K501E, and R544A, reduced the apparent affinity of the high and low affinity ATP effects to different extents

Summary

Introduction

Naϩ/Kϩ-ATPase is an integral membrane protein consisting of ␣ and ␤ subunits and couples ATP hydrolysis to the active transport three Naϩ and two Kϩ ions against their electrochemical gradients. It has been clearly shown that the high affinity ATP binding to the Naϩ-bound enzyme in the presence of Mg2ϩ induced a reversible pyridoxal fluorescence change at position Lys-480 without any ATP-dependent EP formation [10, 12,13,14,15]. The low affinity ATP binding in the presence of Mg2ϩ ϩ Naϩ to the Kϩ-bound enzyme induced a dynamic pyridoxal fluorescence change at Lys-480 and a fluorescein probe at Lys-501 [15]. It is important to understand the relationship between high and low affinity ATP binding for developing a better understanding of the mechanism by which Naϩ/Kϩ-ATPase operates To investigate these points, 10 mutants carrying a single amino acid substitution of the side chain that possibly serve as ligands for ATP in the ATP-binding pocket were constructed. The data suggest that the Arg-544 and possibly Lys-501 are more important for high affinity ATP binding, whereas the Phe-475 and possibly Lys480 are more important for low affinity ATP binding

Methods

Results

Conclusion