Structure and function of ion-channel VO of Na+-translocating V-ATPase

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The role of NtpI C-terminal region of E. hirae Na^+-ATPase was studied. In this region, there is an arginine residue, which is highly conserved among the corresponding subunits of bacterial V-ATPases, at position 573 of NtpI. Substitution of Arg-573 with Glu, Leu, or Gln abolished sodium transport and sodium-stimulated ATP hydrolysis of the enzyme. The conservative replacement of Arg by Lys lowered both activities to about one fifth of those of the wild-type enzyme. We previously reported an ATP-dependent negative cooperativity for Na^+ coupling of this enzyme. This negative cooperativity was weakened by the mutation Arg573Lys; the Hill coefficients for the wild type and mutant enzymes at a saturated ATP concentration were 0.22±0.03 and 0.40±0.05, respectively. The Hill coefficients of both enzymes at limited ATP concentrations approached 1. These results indicate that NtpI Arg573 is indispensable for sodium translocation and for the cooperative features of E. hirae V-ATPase.In addition to Arg-573, the Tyr571, Leu574 and Leu577 were also involved in Na^+ translocation. And other important residues, His-626 and Glu-634, were also required for Na^+-ATPase activity, but their role in ion translocation may differ from conserved His or Glu residues of other a subunits. From these results, it is indicated that the C-terminal region of NtpI is both essential in sodium translocation and stabilization of NtpI.