Reactive Cysteines of the Yeast Plasma-Membrane H+-ATPase (PMA1)

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 plasmamembrane 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 NEMreactive 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 ϳ4-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 pK a 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 ϳ3.5-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.

The Streptococcal Hyaluronan Synthases Are Inhibited by Sulfhydryl-modifying Reagents, but Conserved Cysteine Residues Are Not Essential for Enzyme Function

“…Substrate binding to this site appears to interfere with the reaction between NEM and the nearby Cys residue(s). Similar conclusions about the proximity of Cys residues to substrate binding sites have been reported for several other proteins, including the lactose permease (41), glutathione synthetase (42), glucocorticoid receptor (43), retinoic acid receptor ␤ (44), and plasma membrane proton-ATPase (45). All of these studies found that modification of Cys residues by sulfhydryl reagents decreased the activity of the protein, even though Cys mutagenesis did not inactivate the protein.…”

Evaluation of the antimicrobial activity of ebselen: Role of the yeast plasma membrane H+‐ATPase

“…The physiological consequences of mutating the L-cysteine residues of H + -ATPase have been investigated in several laboratories [39,43,44]. Evidence is available to indicate that single substitution of one of the nine L-cysteine residues in the H + -ATPase of S. cerevisiae SY4 strain with alanine will not affect the expression of this protein, its ATPase activity, or proton extrusion.…”

Effect of thiosulphinates contained in garlic extract on growth, proton fluxes and membrane potential in maize (Zea mays L.) coleoptile segments

“…It is generally agreed that these processes are predominantly regulated by PM H ? -ATPase, which similarly to other ATPases, is sensitive to sulfhydryl reagents (Elzenga et al 1989;Hager and Lanz 1989;Chang and Slayman 1990;Feng and Forgac 1994;Petrov et al 1997;Yang et al 2003).…”