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 (Petrov, 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 plasma-membrane H+ATPase (PMA)1 of Saccharomyces cerevisiae. When membrane vesicles containing the ATPase were incubated with NEM, six of nine mutants with single cysteine substitutions showed sensitivity similar to the wild-type enzyme. By contrast, C221A and C532A were inactivated more slowly than the wild-type control, and the C221, 532A double mutant was completely resistant, indicating that Cys-221 and Cys-532 are NEM-reactive residues. In the presence of 10 mM MgADP, the wild-type ATPase was partially protected against NEM; parallel experiments with the C221A and C532A mutants showed that the protection occurred at Cys-532, located in or near the nucleotide-binding site. Unexpectedly, the inactivation of the C409A ATPase was approximately 4-fold more rapid than in the case of the wild-type enzyme. Experiments with double mutants made it clear that this resulted from an acidic shift in pKa and a consequent acceleration of the reaction rate at Cys-532. One simple interpretation is that substitution of Cys-409 leads to a local conformational change within the central hydrophilic domain. Consistent with this idea, the reaction of fluorescein 5'-isothiocyanate at Lys-474 was also stimulated approximately 3. 5-fold by the C409A mutation. Taken together, the results of this study provide new information about the reactivity of individual Cys residues within the ATPase and pave the way to tag specific sites for structural and functional studies of the enzyme.
Petrov
V V
VV
Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Pardo
J P
JP
Slayman
C W
CW
D002462:Cell Membrane / Q000201:enzymology; D003545:Cysteine / Q000737:chemistry*; D005033:Ethylmaleimide / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D006180:Proton-Translocating ATPases; D003545:Cysteine; D005033:Ethylmaleimide