title: Substrate-decreased modification by diethyl pyrocarbonate of two histidines in isocitrate lyase from Escherichia coli.
abstract: The inactivation of tetrameric 188-kDa isocitrate lyase from Escherichia coli at pH 6.8 (37 degrees C) by diethyl pyrocarbonate, exhibiting saturation kinetics, is accompanied by modification of histidine residues 266 and 306. Substrates isocitrate, glyoxylate, or glyoxylate plus succinate protect the enzyme from inactivation, but succinate alone does not. Removal of the carbethoxy groups from inactivated enzyme by treatment with hydroxylamine restores activity of isocitrate lyase. The present results suggest that the group-specific modifying reagent diethyl pyrocarbonate may be generally useful in determining the position of active site histidine residues in enzymes.
authors.affiliation: Department of Biochemistry and Biophysics, Washington State University, Pullman 99164-4660.
authors.forename: Y H
authors.initials: YH
authors.lastname: Ko
authors.forename: P
authors.initials: P
authors.lastname: Vanni
authors.forename: G R
authors.initials: GR
authors.lastname: Munske
authors.forename: B A
authors.initials: BA
authors.lastname: McFadden
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D004048:Diethylamines / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D006038:Glyoxylates / Q000378:metabolism; D006639:Histidine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007522:Isocitrate Lyase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007523:Isocitrates / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D013056:Spectrophotometry, Ultraviolet; D013386:Succinates / Q000378:metabolism; D019802:Succinic Acid
chemical_list: D004048:Diethylamines; D006038:Glyoxylates; D007523:Isocitrates; D046911:Macromolecular Substances; D013386:Succinates; D006639:Histidine; C034219:isocitric acid; D019802:Succinic Acid; C034281:diethylamine; D007522:Isocitrate Lyase; C031150:glyoxylic acid; D004047:Diethyl Pyrocarbonate; C005229:itaconic acid