A Hydrophobic Microenvironment Significantly Influences the Reactivity of the Catalytically Relevant Thiols of the Na+/K+-ATPase

The transmembrane protein responsible for the electrogenic transport of Na+ and K+ across the plasma membrane, the Na+/K+-ATPase, highly vulnerable redox modulations and thiol modifying agents due to the presence of thiol groups at the nucleotide and cationic sites. However, reports have demonstrated a preferential interaction of these protein thiols with oxidizing agents. The reactivity of protein thiols is strongly linked with the nature of the microenvironment of these thiols, hence, the present study sought to experimentally elucidate key features of the microenvironment of the catalytically relevant thiols at the substrate-binding sites of this crucial enzyme. Two thiol modifiers with similar thiol-reactive mechanism, but different molecular properties, iodoacetamide (IA) and N-acetyl-4-phenyliodoacetamide (APIAM), were employed. It was observed that while both compounds demonstrated excellent thiol-oxidizing properties in the chemical model, only APIAM had an inhibitory effect on the activity of the Na+/K+-ATPase. The involvement of the catalytically relevant thiols at the nucleotide and cation-binding sites of the enzyme in APIAM-mediated inhibition was confirmed by the protective effect of preincubating the reaction system with dithiothreitol (DTT). The findings from this study suggest that the catalytically relevant thiols of this enzyme are likely buried in a hydrophobic microenvironment. This could be a part of the protective measure of nature for these vulnerable protein thiols. Further details from our findings can be explored in the therapeutic management of diseases for which a dysfunction in the Na+/K+-ATPase have been identified. HighlightsO_LIThe transmembrane Na+/K+-ATPase has well-defined substrate-binding domains exposed to both aqueous microenvironment and buried within the hydrophobic transmembrane microenvironment C_LIO_LIThese microenvironments influence vulnerability of the critical thiols of the enzyme to oxidative assault C_LIO_LIThese thiols are likely buried in the hydrophobic core of the enzyme, thus selecting its susceptibility to thiol modfying agents C_LI