Jm. Arguello et al., Functional role of oxygen-containing residues in the fifth transmembrane segment of the Na,K-ATPase alpha subunit, ARCH BIOCH, 364(2), 1999, pp. 254-263
The functional roles of Tyr771, Thr772, and Asn776 in the fifth transmembra
ne segment of the Na, ATPase a subunit were studied using site-directed mut
agenesis, expression, and kinetics analysis. Nonconservative replacements T
hr772Tyr and Asn776Ala led to reduced Na,K-ATPase turnover, Replacements at
these positions (Asn776Ala, Thr772Leu, and Thr772Tyr) also led to high Na-
ATPase activity tin the absence of K+). However, Thr772- and Asn776-substit
uted enzymes showed only small alterations in the apparent Na+ and K+ affin
ities (K-1/2 for Na,K-ATPase activation). Thus, the high Na-ATPase activity
does not appear related to cation-binding alterations. It is probably asso
ciated with conformational alterations which lead to an acceleration of enz
yme dephosphorylation by Naf acting at the extracellular space (Arguello et
al. J. Biol. Chem. 271, 24610-24616, 1996), Nonconservative substitutions
at position 771 (Tyr771Ala and Tyr771Ser) produced a significant decrease o
f enzyme turnover. Enzyme-Na+ interaction was greatly changed in these enzy
mes, while their activation by K+ did not appear affected Although the Nat
It, for Na,K-ATPase stimulation was unchanged (Tyr771Ala, Tyr771Ser), the a
ctivation by this cation showed no cooperativity (Tyr771Ala, n(Hill) = 0.75
; Tyr771Ser, n(Hill) = 0.92; Control, n(Hill) = 2.28). Substitution Tyr771P
he did not lead to a significant reduction in the cooperativity of the ATPa
se Na+ dependence (n(Hill) = 1.91). All Tyr771-substituted enzymes showed l
ow steady-state levels of phosphoenzyme during Na-activated phosphorylation
by ATP, Phosphorylation levels were not increased by oligomycin, although
the drug bound and inactivated Tyr771-substituted enzymes. No E1 <----> E2
equilibrium alterations were detected using inhibition by vanadate as a pro
be. The data suggest that Tyr771 might play a central role in Na+ binding a
nd occlusion without participating in K+-enzyme interactions. (C) 1999 Acad
emic Press.