title: The H385N mutant of 5-enolpyruvylshikimate-3-phosphate synthase: kinetics, fluorescence, and nuclear magnetic resonance studies.
abstract: The site-directed mutagenesis of histidine-385 of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase is reported. The H385N mutant is compared with wild type by a number of methods. H385N was found to retain 6% activity. Kinetic parameters, including Km values for the natural substrates and Ki and Kd values for the inhibitor glyphosate, were found to be similar to wild type. Unlike wild-type enzyme, H385N EPSP synthase does not show accumulation of enzyme-bound product (EPSP) in the 13C NMR spectrum under equilibrium conditions. These results suggest that this H385N mutant is less catalytically competent than the previously studied H385Q mutant and that the N(epsilon)H of histidine may be involved in hydrogen bonding to another residue involved in complexing the substrates/ products.
authors.affiliation: Department of Biochemistry and Biophysics, Washington State University, Pullman 99164-4660, USA.
authors.forename: W A
authors.initials: WA
authors.lastname: Shuttleworth
authors.forename: J N
authors.initials: JN
authors.lastname: Evans
mesh_terms: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014166:Transferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D006639:Histidine; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase