title: [Production and catalytic properties of point mutants Phe41--->His and Phe143--->Glu of horseradish peroxidase, expressed in Escherichia coli].
abstract: Recombinant horseradish peroxidase and its single-point mutants, F4IH and H143E, have been reactivated from E. coli inclusion bodies with a 25% yield. Both mutations affect heme entrapment as well as enzyme stability and activity. A more than 40-fold decrease in the specific activity towards ABTS is associated with different steps of peroxidase catalysis. F41H replacement results in a drop of both rate constants by two and one orders or magnitude for hydrogen peroxide and ABTS, respectively. The mechanism of iodide oxidation by the F41H mutant fits into a ternary interaction. The F143E replacement mainly affects the steps of ABTS oxidation and product dissociation. It is suggested that the role of replaced phenylalanine residues consists in the formation of a highly hydrophobic pocket allowing for strong non-covalent binding of the heme porphyrin ring.
authors.forename: I G
authors.initials: IG
authors.lastname: Gazarian
authors.forename: V V
authors.initials: VV
authors.lastname: Doseeva
authors.forename: A G
authors.initials: AG
authors.lastname: Galkin
authors.forename: V I
authors.initials: VI
authors.lastname: Tishkov
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics*; D006418:Heme / Q000378:metabolism; D006639:Histidine / Q000235:genetics*; D006735:Horseradish Peroxidase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000235:genetics*; D017354:Point Mutation
chemical_list: D004274:DNA, Recombinant; D018698:Glutamic Acid; D006418:Heme; D010649:Phenylalanine; D006639:Histidine; D006735:Horseradish Peroxidase
vernacular_title: Poluchenie i kataliticheskie svoĭstva tochechnykh mutantov Phe41--->His i Phe143--->Glu peroksidazy khrena, ékspressiruemykh v Escherichia coli.