title: Wild-type and mutant forms of recombinant horseradish peroxidase C expressed in Escherichia coli. Substrate specificity and stability under irradiation.
abstract: Two horseradish peroxidase C (HRPC) mutants with substitutions in the active center, i.e., Phe41-->His and Phe143-->Glu, were compared to the wild-type recombinant enzyme expressed in Escherichia coli in terms of the enzymatic activity and stability under irradiation. Both mutations caused a significant decrease in activity, but it was still possible to follow the effect of mutations on the key steps of the reaction mechanism. Phe41 can be considered a nonpolar barrier separating histidine residues in the active center and providing a firm noncovalent binding with the highly hydrophobic porphyrin ring. The replacement of Phe41 with the ionizable His residue destabilizes the enzyme. The Phe143-->Glu replacement creates a negative charge at the entrance of the heme-binding pocket, and protects the latter from both donor substrates and free radicals. The radiolytic inactivation of the wild-type and mutant forms of recombinant HRP suggested different binding sites for iodide, 2,2'-bis(3-ethylbenzothiasoline-6-sulfonate (ABTS), guaiacol, and O-phenylene diamine. The study of kinetics and inactivation is in agreement with the direct binding of iodide to the heme porphyrin ring. The results also suggest that the ABTS binding site is less accessible than that for O-phenylene diamine.
authors.affiliation: Chemical Faculty of Moscow State University, GSP, Russia.
authors.forename: E A
authors.initials: EA
authors.lastname: Mareeva
authors.forename: M A
authors.initials: MA
authors.lastname: Orlova
authors.forename: V V
authors.initials: VV
authors.lastname: Doseeva
authors.forename: D B
authors.initials: DB
authors.lastname: Loginov
authors.forename: A G
authors.initials: AG
authors.lastname: Galkin
authors.forename: I G
authors.initials: IG
authors.lastname: Gazarian
authors.forename: V I
authors.initials: VI
authors.lastname: Tishkov
mesh_terms: D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006735:Horseradish Peroxidase / Q000235:genetics*; D007527:Isoenzymes / Q000235:genetics*; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D007527:Isoenzymes; D011994:Recombinant Proteins; D006735:Horseradish Peroxidase