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G. Yu. Lomakina, Yu. A. Modestova, N. N. Ugarova
The thermostability enhancement of firefly luciferase Luciola
mingrelica by site-directed mutagenesis of non-concervative residies Cys62
and Cys146
Abstract
Mutant forms of L.mingrelica firefly luciferase with
point mutations Cys62Ser and Cys146Ser were prepared by site-directed
mutagenesis. Mutations were not influenced the catalytic and spectral
properties of the enzyme. Fast (k1)
and slow (k2) inactivation rate constants at 37°C for wild type enzyme and its mutants with or without
DTT were determined. Several times increase of mutant forms thermostability on
both stages of the inactivation was shown. For wild type enzyme three–fold
decrease of k2 value
in the presence of DTT was shown while k1 and k2 values for mutant forms were not changed. Thus it was concluded the significance of
Cys62 and Cys146 residues in the L.mingrelica firefly luciferase
inactivation process and their
replacements to Ser resulted in the enzyme stabilization.
Copyright (C) Chemistry Dept., Moscow State University, 2002
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