FUNCTIONAL CONSEQUENCES OF SUBSTITUTIONS OF THE CARBOXYL RESIDUE GLUTAMATE-779 OF THE NA,K-ATPASE

Carboxyl-containing amino acids in the transmembrane segments appear t o be important for sodium- and potassium-activated adenosinetriphospha tase (Na,K-ATPase) activity, Substitution of Glu779 with Leu in a ouab ain-resistant isoform inactivates the overall enzyme activity (Jewell- Motz and Lingrel, 1993). Chemical modification of this residue results in inactivation of Na,K-ATPase in a Na+ and K+ protectable manner (Ar guello and Kaplan, 1991, 1994). These experiments suggest that this re sidue is important in cation binding. To further understand the role o f Glu779 in Na,K-ATPase function, we have substituted this with four a mino acids (Gin, Asp, Ala, and Leu) using site-directed mutagenesis co upled with expression and characterized the expressed enzyme. The amin o acid substitutions were introduced into a modified sheep RD alpha 1 isoform that is relatively resistant to this drug. Enzyme carrying the E779Q and E779A replacements conferred ouabain resistance to the sens itive HeLa cells, while expression of enzyme carrying the E779D and E7 79L substitutions did not. Further analysis of isolated plasma membran es containing altered enzymes E779Q and E779A confirmed that they reta in Na,K-ATPase activity. Analysis of cation stimulation of Na,K-ATPase activity revealed that the E779Q substituted enzyme exhibited a simil ar apparent affinity for K+ and a 2.6-fold decrease in affinity for Na + compared with control enzyme. The E779A replacement caused a 6.6-fol d and 5-fold decrease in apparent affinity for K+ and Na+, respectivel y. There is no difference in apparent affinity for ATP at the low affi nity site for either E779Q or E779A. Moreover, both E779Q and E779A ar e phosphorylated by ATP, and their turnover numbers are not significan tly different from that of control enzyme. These results suggest that Glu779 may be important for cation interactions with the Na,K-ATPase; however, the carboxyl side chain is not required for normal enzyme tur nover.