Production and catalytic properties of Phe41->His and Phe143->Glu single-point mutants of horseradish peroxidase expressed in E-coliстатья
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Дата последнего поиска статьи во внешних источниках: 27 мая 2015 г.
Местоположение издательства:Road Town, United Kingdom
Первая страница:1187
Последняя страница:1192
Аннотация:The wild-type recombinant horseradish peroxidase and its single-point mutants Phe41 --> His and Phe143 --> Glu have been reactivated from E. coli inclusion bodies with 25% yield. Both mutations affect the entrapment of heme by the apoenzyme and the enzyme stability and activity. The replacements result in a 40-fold decrease in the specific activity, and, according to steady-state kinetic studies, they affect different steps in the reaction mechanism. The F41H replacement results in a drop in rate constants toward both hydrogen peroxide and ABTS, by two and one order of magnitude, respectively. In the case of iodide oxidation the catalytic mechanism is changed and the third order rare constant is equal to 2.5 . 10(5) M(-2). sec(-1). The F143E replacement mainly affects ABTS oxidation and its product dissociation steps. In the reaction of iodide oxidation the mutant exhibits an order lower rate constants both for iodide and hydrogen peroxide. The role of the replaced phenylalanine residues may consist in the formation of a highly hydrophobic pocket providing for firm noncovalent binding of the heme porphyrin ring.