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A. A. Poloznikov, P. A. Savitsky, D. M. Khushpulyan, T. A. Chubar, I. G. Gazaryan, V. I. Tishkov

Preparation and properties of mutant tobacco peroxidase with additional tryptophan residues

Abstract

Mutant forms of tobacco peroxidase Gln116Trp and Leu157Trp have been prepared and expressed in E. coli cells BL21(DE3) CodonPlus. The muteins were expressed at the level up to 40% of total E.coli proteins as inclusion bodies. Both mutants were prepared inactive form according to standard refolding procedure optimized for wild-type TOP. Yield of active proteins was about 9% and specific activity with ABTS 20003000 U per mf of protein. Kinetoc properties of mutant enzymes in reaction of ABTS oxidation with hydrogenperoxide have been studied. It was shown that reaction proceeds through ping-pong kinetic mechanism but rate constants for separate stages were different for mutants. It was supposed that additional tryptophane residues participate in enzyme catalysis.
Moscow University Chemistry Bulletin.
2006, Vol. 47, No. 1, P. 20
   

Copyright (C) Chemistry Dept., Moscow State University, 2002
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