Abstract
Mutant forms of the firefly (Luciola mingrela) luciferase with point mutations Cys62Ser and Cys146Ser were obtained by site-directed mutagenesis. The mutations did not affect the catalytic activity and fluorescence spectra of the enzyme. The rate constants of the fast (k 1) and slow (k 2) stages of thermoinactivation of the wild-type and mutant enzymes were determined at 37°C in the absence and presence of 12 mM dithiothreitol (DTT). The thermostability of the mutant forms of luciferase increased several times compared to the wild-type enzyme. In the presence of DTT, k 2 of the wild-type enzyme decreased three times whereas neither k 1 nor k 2 of the mutant forms changed. It was concluded that amino acid residues Cys62 and Cys146 play a major role in luciferase inactivation and that their substitution with Ser stabilizes the enzyme.
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Original Russian Text © G.Yu. Lomakina, Yu.A. Modestova, N.N. Ugarova, 2008, published in Vestnik Moskovskogo Universiteta. Khimiya, 2008, No. 2, pp. 81–85.
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Lomakina, G.Y., Modestova, Y.A. & Ugarova, N.N. Enhancement of thermostability of the Luciola mingrelica firefly luciferase by site-directed mutagenesis of nonconservative cysteine residues Cys62 and Cys146. Moscow Univ. Chem. Bull. 63, 63–66 (2008). https://doi.org/10.3103/S002713140802003X
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DOI: https://doi.org/10.3103/S002713140802003X