Reactivity of cysteines in the transmembrane region of the Na, K-ATPase alpha subunit probed with Hg(2+).
To gain insight into the structure and conformational coupling in the Na,K-ATPase, this study characterized the reaction of the alpha1 subunit transmembrane cysteines with a small probe. Intact HeLa cells expressing heterologous Na,K-ATPase were treated with (microm) HgCl(2) after placing the enzyme predominantly in either of two conformations, phosphorylated E2P.Na/E2P or dephosphorylated ATP.E1. K/ATP.E1. Under both conditions the treatment led to enzyme inactivation following a double exponential kinetic as determined by ouabain-sensitive K(+) uptake measurements. However, the rate constant of the slow reacting component was ten times larger when the protein was probed in a medium that would favor enzyme phosphorylation. Enzymes carrying mutations of cysteines located in the alpha1 subunit transmembrane region were used to identify the reacting-SH groups. Replacement Cys104Ser reduced enzyme inactivation by removing the slow reacting component under both treatment conditions. Replacement of Cys964 reduced the inactivation rate constant of the fast reacting component (79%) and removed the slow reacting component when the dephosphorylated enzyme was treated with Hg(2+). Moreover, Cys964Ser substituted enzyme was insensitive to Hg(2+) when treated under phosphorylation conditions. These results indicate that Cys964 is involved in the fast inactivation by Hg(2+). Although the double mutant Cys964, 104Ser was still partially inactivated by treatment under nonphosphorylating conditions, an enzyme devoid of transmembrane cysteines was insensitive to Hg(2+) under all treatment conditions. Thus, this enzyme provides a background where accessibility of engineered transmembrane cysteines can be tested.
Zichittella
A E
AE
Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, MA 01609, USA.
Shi
H G
HG
Argüello
J M
JM
D002462:Cell Membrane / Q000201:enzymology; D003545:Cysteine / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008628:Mercury / Q000494:pharmacology; D015335:Molecular Probes; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011487:Protein Conformation; D021122:Protein Subunits; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics
D004791:Enzyme Inhibitors; D015335:Molecular Probes; D021122:Protein Subunits; D000254:Sodium-Potassium-Exchanging ATPase; D008628:Mercury; D003545:Cysteine