title: Reactivity of the H(+)-ATPase from Kluyveromyces lactis to sulfhydryl reagents.
abstract: N-Ethylmaleimide (NEM) inhibited the H(+)-ATPase (EC 3.6.1.35) from Kluyveromyces lactis with a second-rate constant of 200 M-1 min-1. H(+)-ATPase was partially protected by Mg-ADP. Low concentrations of Mg protected ATPase from the effects of NEM, while high Mg sensitized ATPase to NEM. The reaction of 14C-NEM with the native enzyme modified three cysteine residues/monomer, two of which were involved in 80% of the inactivation of the enzyme. In the presence of Mg-ADP, NEM binding to the first residue had only a slight effect on the activity (10-20% inhibition). After further incubation, the modification of a second cysteine residue (probably cys-221) inactivated the ATPase. Methyl methanethiosulfonate did not inhibit the H(+)-ATPase but resulted in a NEM-resistant H(+)-ATPase. There seems to be at least one cys (probably cys-532) at, or near, the nucleotide binding site of the H(+)-ATPase, which does not appear to be essential for activity. Modification of a second cys residue (cys-221) also resulted in inactivation by NEM; this residue was not protected by ADP and thus probably is not at the ATP binding site.
authors.affiliation: Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México, D.F.
authors.forename: G
authors.initials: G
authors.lastname: Guerra
authors.forename: S
authors.initials: S
authors.lastname: Uribe
authors.forename: J P
authors.initials: JP
authors.lastname: Pardo
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D003545:Cysteine; D005033:Ethylmaleimide / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D007716:Kluyveromyces / Q000201:enzymology*; D008278:Magnesium Sulfate / Q000494:pharmacology; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives* / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification
chemical_list: C014674:methyl methanethiosulfonate; D000244:Adenosine Diphosphate; D008278:Magnesium Sulfate; D008741:Methyl Methanesulfonate; D006180:Proton-Translocating ATPases; D003545:Cysteine; D005033:Ethylmaleimide