Jan 17, 1997

Reactive cysteines of the yeast plasma-membrane H+-ATPase (PMA1). Mapping the sites of inactivation by N-ethylmaleimide

The Journal of Biological Chemistry
V V PetrovC W Slayman

Abstract

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 lo...Continue Reading

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Mentioned in this Paper

Ethylmaleimide
Saccharomyces cerevisiae allergenic extract
Adenosine Triphosphatases
Isothiocyanates
Mutagenesis, Site-Directed
Macromolecular Alteration
Fluorescein
Vesicle
Ligand Binding Domain
Cysteine

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