title: Localization of the active site of type II dehydroquinases. Identification of a common arginine-containing motif in the two classes of dehydroquinases.
abstract: A novel method based on electrospray mass spectrometry (Krell, T., Pitt, A. R., and Coggins, J. R. (1995) FEBS Lett. 360, 93-96) has been used to localize active site residues in the type I and type II dehydroquinases. Both enzymes have essential hyper-reactive arginine residues, and the type II enzymes have an essential tyrosine residue. The essential hyper-reactive Arg-23 of the Streptomyces coelicolor type II enzyme has been replaced by lysine, glutamine, and alanine residues. The mutant enzymes were purified and shown by CD spectroscopy to be structurally similar to the wild-type enzyme. All three mutant enzymes were much less active, for example the kcat of the R23A mutant was 30,000-fold reduced. The mutants all had reduced Km values, indicating stronger substrate binding, which was confirmed by isothermal titration calorimetry experiments. A role for Arg-23 in the stabilization of a carbanion intermediate is proposed. Comparison of the amino acid sequence around the hyper-reactive arginine residues of the two classes of enzymes indicates that there is a conserved structural motif that might reflect a common substrate binding fold at the active center of these two classes of enzyme.
authors.affiliation: Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Scotland, United Kingdom.
authors.forename: T
authors.initials: T
authors.lastname: Krell
authors.forename: M J
authors.initials: MJ
authors.lastname: Horsburgh
authors.forename: A
authors.initials: A
authors.lastname: Cooper
authors.forename: S M
authors.initials: SM
authors.lastname: Kelly
authors.forename: J R
authors.initials: JR
authors.lastname: Coggins
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics*; D001665:Binding Sites; D002151:Calorimetry; D002942:Circular Dichroism; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D013302:Streptomyces / Q000201:enzymology
chemical_list: D001120:Arginine; D006836:Hydro-Lyases; C020000:3-dehydroquinate dehydratase