title: Vanadate-dependent photomodification of serine 319 and 321 in the active site of isocitrate lyase from Escherichia coli.
abstract: Vanadate was used as a substrate analogue to modify and subsequently localize active site serine residues of isocitrate lyase from Escherichia coli. Irradiation of the enzyme on ice with UV light in the presence of vanadate resulted in inactivation. Inactivation was prevented by the substrates glyoxylate or Ds-isocitrate and to a much lesser extent by succinate. Reduction of photoinactivated isocitrate lyase by NaBH4 partially restored enzyme activity. The photomodified enzyme was labeled by reduction with NaB[3H]4 in the presence and absence of the substrates succinate plus glyoxylate. Highly differential labeling of serine residues 319 and 321 in the absence of substrates suggests their importance in the action of isocitrate lyase. These residues are highly conserved in all five known sequences of this enzyme.
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: C R
authors.initials: CR
authors.lastname: Cremo
authors.forename: B A
authors.initials: BA
authors.lastname: McFadden
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D006038:Glyoxylates / Q000378:metabolism; D006868:Hydrolysis; D007522:Isocitrate Lyase / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D007523:Isocitrates / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D010777:Photochemistry; D016415:Sequence Alignment; D012694:Serine / Q000378:metabolism*; D013379:Substrate Specificity; D014357:Trypsin; D014466:Ultraviolet Rays; D014638:Vanadates / Q000378:metabolism*
chemical_list: D006038:Glyoxylates; D007523:Isocitrates; D014638:Vanadates; D012694:Serine; C034219:isocitric acid; D014357:Trypsin; D007522:Isocitrate Lyase; C031150:glyoxylic acid