title: [Physicochemical properties of recombinant luciferase from the firefly Luciola mingrelica and its mutant forms].
abstract: Physico-chemical properties of the recombinant L. mingrelica luciferase synthesized by E. coli cells have been studied. The catalytic and spectral properties of recombinant luciferase were similar to those of the native enzyme but the former was less stable in the presence of the additional Cys residue. The mutant forms of L. mingrelica firefly luciferase with point mutations Cys-82-->Ala, Cys-260-->Ala, Cys-393-->Ala and Thr-204-->Asp, have been constructed using the method of site-specific mutagenesis. Mutations Cys-82,260,393-->Ala changed slightly the Km values for ATP and luciferin but did not influence kcat. The Cys-393-->Ala mutant appeared to be more stable in comparison with the native enzyme. Mutation Thr-204-->Asp resulted in a 8-fold increase in the ATP binding constant and in a 2-fold increase in the kcat, thus indicating that Thr-204 may be located in the ATP-binding region of luciferase. Dithiothreitol, ethylene glycol, bovine serum albumin and trehalose had a stabilizing effect on the native, recombinant and mutant luciferases.
authors.affiliation: Faculty of Chemistry, M.V. Lomonosov Moscow State University.
authors.forename: E I
authors.initials: EI
authors.lastname: Dement'eva
authors.forename: E E
authors.initials: EE
authors.lastname: Zheleznova
authors.forename: G D
authors.initials: GD
authors.lastname: Kutuzova
authors.forename: I A
authors.initials: IA
authors.lastname: Lundovskikh
authors.forename: N N
authors.initials: NN
authors.lastname: Ugarova
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D001517:Coleoptera / Q000201:enzymology*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D008156:Luciferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D017354:Point Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D008156:Luciferases
vernacular_title: Fiziko-khimicheskie svoĭstva rekombinantnoĭ liutsiferezy svetliakov Luciola mingrelica i eë mutantnykh form.