REACTIVE CYSTEINES OF THE YEAST PLASMA-MEMBRANE H-ATPASE (PMA1) - MAPPING THE SITES OF INACTIVATION BY N-ETHYLMALEIMIDE()

We have taken advantage of cysteine mutants described previously (Petr ov, V. V., and Slayman, C. W. (1995) J. Biol. Chem. 270, 28535-28540) to map the sites at which N-ethylmaleimide (NEM) reacts with the plasm amembrane H+ ATPase (PMA)1 of Saccharomyces cerevisiae. When membrane vesicles containing the ATPase were incubated with NEM, six of nine mu tants with single cysteine substitutions showed sensitivity similar to the wild type enzyme. By contrast, C221A and C532A were inactivated m ore slowly than the wild-type control, and the C221, 532A double mutan t was completely resistant, indicating that Cys-221 and Cys-532 are NE M-reactive residues. In the presence of 10 mM MgADP, the wild-type ATP ase was partially protected against NEM; parallel experiments with the C221A and C532A mutants showed that the protection occurred at Cys-53 2, located in or near the nucleotide-binding site. Unexpectedly, the i nactivation of the C409A ATPase was similar to 4-fold more rapid than in the case of the wild-type enzyme. Experiments with double mutants m ade it clear that this resulted from an acidic shift in pK(alpha) and a consequent acceleration of the reaction rate at Cys-532. One simple interpretation is that substitution of Cys-409 leads to a local confor mational change within the central hydrophilic domain. Consistent with this idea, the reaction of fluorescein 5'-isothiocyanate at Lys-474 w as also stimulated similar to 3.5-fold by the C409A mutation. Taken to gether, the results of this study provide new information about the re activity of individual Cys residues within the ATPase and pave the way to tag specific sites for structural and functional studies of the en zyme.