[Physicochemical properties of recombinant luciferase from the firefly Luciola mingrelica and its mutant forms].

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. -->
Substance Nomenclature: 0 (Recombinant Proteins)
8L70Q75FXE (Adenosine Triphosphate)
EC 1.13.12.- (Luciferases)
Entry Date(s): Date Created: 19960101 Date Completed: 19960816 Latest Revision: 20171116
Update Code: 20221213
PMID: 8679773
Autor: Dement'eva EI; Faculty of Chemistry, M.V. Lomonosov Moscow State University., Zheleznova EE, Kutuzova GD, Lundovskikh IA, Ugarova NN
Jazyk: ruština
Zdroj: Biokhimiia (Moscow, Russia) [Biokhimiia] 1996 Jan; Vol. 61 (1), pp. 152-9.
Abstrakt: 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.
Databáze: MEDLINE
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