The structural-functional roles of 23 cysteines present in the sheep (Na, K)-ATPase α1 subunit were studied using site directed mutagenesis, expression, and kinetics analysis. Twenty of these cysteines were individually substituted by alanine or serine. Cys452, Cys455 and Cys456 were simultaneously replaced by serine. These substitutions were introduced into an ouabain resistant α1 sheep isoform and expressed in HeLa cells under ouabain selective pressure. HeLa cells transfected with a cDNA encoding for replacements of Cys242 did not survive ouabain selective pressure. Single substitutions of the remaining cysteines yielded functional enzymes, although some had reduced turnover rates. Only minor variations were observed in the enzyme Na+ and K+ dependence as a result of these replacements. Some substitutions apparently affect the E1 mutually implies E2 equilibrium as suggested by changes in the K(m) of ATP acting at its low affinity binding site. These results indicate that individual cysteines, with the exception of Cys242, are not essential for enzyme function. Furthermore, this suggests that the presence of putative disulfide bridges is not required for α1 subunit folding and subsequent activity. A (Na, K)-ATPase lacking cysteine residues in the transmembrane region was constructed (Cys104, 138, 336, 802, 911, 930, 964, 983Xxx). No alteration in the K(1/2) of Na+ or K+ for (Na, K)-ATPase activation was observed in the resulting enzyme, although it showed a 50% reduction in turnover rate. ATP binding at the high affinity site was not affected. However, a displacement in the E1 mutually implies E2 equilibrium toward the E1 form was indicated by a small decrease in the K(m) of ATP at the low affinity site accompanied by an increase in IC50 for vanadate inhibition. Thus, the transmembrane cysteine-deficient (Na, K)-ATPase appears functional with no critical alteration in its interactions with physiological ligands. © 2000 Elsevier Science B.V.
Shi, H. G., Mikhaylova, L., Zichittella, A. E., & Argüello, J. M. (2000). Functional role of cysteine residues in the (Na, K)-ATPase α subunit. Biochimica et Biophysica Acta - Biomembranes, 1464(2), 177–187. https://doi.org/10.1016/S0005-2736(99)00245-X