title: ATP-dependent inactivation of the beta-Ser339Cys mutant F1-ATPase from Escherichia coli by N-ethylmaleimide.
abstract: We introduced mutations at the highly-conserved residue Ser-339 in subunit beta of Escherichia coli F1-ATPase. The mutations beta S339Y and beta S339F abolished ATPase activity and impaired enzyme assembly. In contrast beta S339C F1 retained function to a substantial degree. N-Ethylmaleimide (NEM) at 0.2-0.3 mM inactivated beta S339C F1-ATPase by 80-95% in the presence of MgATP or MgADP but did not inactivate appreciably in absence of nucleotide or presence of EDTA. In absence of nucleotide, 0.7 mol of [14C-NEM] was incorporated into beta-subunits of 1.0 mol F1: in presence of MgATP the amount was 1.7 mol/mol, i.e. the introduced Cys residue became more accessible to reaction in the presence of MgATP. In the X-ray structure of F1 (Abrahams et al. (1994) Nature 370, 621-628) one of the catalytic nucleotide-binding domains is empty (on the "beta E subunit") and contains a cleft. Residue beta-339 lies within this cleft; the cleft does not occur in the other two beta-subunits. Our data are consistent with the conclusion that in wild-type enzyme under physiological conditions, MgATP or MgADP induce an enzyme conformation in which residue beta-Ser-339 becomes more exposed, possibly similar to the situation seen in the "beta E-subunit" in the X-ray structure.
authors.affiliation: Department of Biochemistry, University of Rochester Medical Center, New York 14642, USA.
authors.forename: G
authors.initials: G
authors.lastname: Schmidt
authors.forename: A E
authors.initials: AE
authors.lastname: Senior
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D001483:Base Sequence; D003545:Cysteine / Q000235:genetics; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005033:Ethylmaleimide / Q000378:metabolism / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000235:genetics; D012694:Serine / Q000235:genetics; D013329:Structure-Activity Relationship
chemical_list: D012694:Serine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D006180:Proton-Translocating ATPases; D003545:Cysteine; D005033:Ethylmaleimide