A diagnostic feature of P-ATPases is a phosphorylation motif (DKTGTLT), located in the hydrophilic center of the polypeptide chain, within which the beta-aspartyl-phosphate reaction intermediate is formed. The roles of four invariant residues (Lys379, Thr380, Thr382, and Thr384) in this region of the yeast plasma membrane H(+)-ATPase have been analyzed by site-directed mutagenesis. In addition, a set of six insertion mutants was generated containing a single glycine residue at each of the indicated sites: [sequence: see text] C S D K T GT LT to examine spatial arrangements within this highly conserved domain. In order to minimize toxic effects of the mutations on cell growth, the defective ATPases were expressed behind an inducible heat shock promoter and targeted to an intracellular pool of secretory vesicles, while wild-type ATPase was maintained in the plasma membrane where it is required for viability. Secretory vesicles containing mutant ATPase were isolated as described previously (Nakamoto, R. K., Rao, R., and Slayman, C. W. (1991) J. Biol. Chem. 266, 7940-7949) and assayed for the amount of ATPase polypeptide and for rates of ATP hydrolysis and H+ pumping. All of the insertion mutations led to biosynthetic arrest of the defective enzyme, with no ATPase appearing in the secretory vesicles. Nonconservative amino acid substitutions (Lys-->Gln, Thr-->Ala) inactivated the ATPase, whereas conservative substitutions (Lys-->Arg, Thr-->Ser) retained partial activity which has been characterized in detail. There was little or no change in the Km for ATP or the pH optimum in any of the mutant enzymes. Strikingly, however, all displayed an increase in resistance to vanadate, consistent with the idea that the residues in question contribute to a phosphate/vanadate binding site or that they affect the equilibrium between E1 and E2 conformations of the enzyme.

• PMID: 8454643
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Reference Type

Journal Article | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, P.H.S.

Authors

Rao R, Slayman CW

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