ABSTRACT
Heme-containing plant peroxidases (EC 1.11.1.7) contain a highly conserved single tryptophan residue. Its replacement with Phe in recombinant horseradish peroxidase (rHRP) increased the stability of the mutant enzyme in acid media. The kinetic properties of native, wild-type, and W117F mutant recombinant horseradish peroxidase in the reactions of ammonium 2, 2;-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), guaiacol, and o-phenylenediamine oxidation are very similar. However, significant changes in the reaction rate constant characteristic for the monomolecular rate-limiting step ascribed either to product dissociation from its complex with the enzyme or electron transfer from the substrate to the active site within the Michaelis complex were observed. The data indirectly indicate the participation of the single Trp residue in oxidation of ABTS and guaiacol and possible differences in kinetic mechanisms for oxidation of ABTS, guaiacol, and o-phenylenediamine.
Subject(s)
Horseradish Peroxidase/metabolism , Tryptophan/chemistry , Catalysis , Enzyme Stability , Horseradish Peroxidase/chemistry , Hydrogen-Ion Concentration , Kinetics , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
The tryptophanless mutant of horseradish peroxidase, W117F, has been constructed and expressed in Escherichia coli. The mutation affects enzyme folding and stability. The optimum composition of the refolding medium requires the presence of ammonium sulfate. The yield of mutant is ca. 8000 U per liter of the optimized refolding medium with the specific activity of 1100-1500 U/mg (compared to 25, 000 U per liter and 2000 U/mg for the recombinant wild-type enzyme). The mutant is more stable in acid media, in the reaction course and toward irradiation. The effect of hydrogen peroxide pretreatment on radiation-induced inactivation of the wild-type and mutant enzyme indirectly indicates participation of Trp-117 in electron transfer pathways through the enzyme molecule. This is in agreement with the steady-state kinetic data interpreted in terms of Trp-117 participation in electron transfer within the Michaelis complex.
Subject(s)
Amino Acid Substitution , Horseradish Peroxidase/metabolism , Tryptophan/chemistry , Amino Acid Sequence , Catalysis , Electrons , Enzyme Activation/radiation effects , Enzyme Stability/radiation effects , Escherichia coli/genetics , Horseradish Peroxidase/chemistry , Horseradish Peroxidase/genetics , Horseradish Peroxidase/isolation & purification , Hydrogen Peroxide/metabolism , Hydrogen-Ion Concentration , Inclusion Bodies , Luminescent Measurements , Models, Molecular , Molecular Sequence Data , Oxidation-Reduction , Plants/enzymology , Protein Folding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Solubility , Tryptophan/geneticsABSTRACT
The effect of extremely acidic pH on the stability of tobacco peroxidase and lignin peroxidase holoenzymes has been studied. Stabilization of tobacco peroxidase holoenzyme in the presence of calcium cations at pH < 2 and stabilization of lignin peroxidase at pH > 2 in the presence of veratryl alcohol have been shown. The dependence of the reaction rate constant for hydrogen peroxide interaction with tobacco peroxidase on pH suggests that the reaction rate is under control of a group with pK of 2.5. A tobacco peroxidase model structure has been created by means of homology modeling on the basis of the tobacco peroxidase sequence and the coordinates of peanut peroxidase crystal structure. The model structure demonstrates the presence of the negatively charged Glu-141 at the entrance to the active site and its electrostatic repulsion from heme propionates and triad of Asp-76, -79, and -80 residues. The results on tobacco holoperoxidase stabilization at pH 1.8 in the presence of calcium cations and drop in reaction rate constant for the enzyme interaction with hydrogen peroxide are explained by a hypothetical formation of ionic bonds between Glu-141 and the triad of aspartic acid residues via calcium cation lowering the accessibility of the active site and stabilizing the holoenzyme.
Subject(s)
Nicotiana/enzymology , Peroxidase/metabolism , Plants, Toxic , Amino Acid Sequence , Arachis/enzymology , Arachis/genetics , Calcium/metabolism , Catalytic Domain , Enzyme Stability , Holoenzymes/chemistry , Holoenzymes/genetics , Holoenzymes/metabolism , Hydrogen Peroxide/metabolism , Hydrogen-Ion Concentration , Kinetics , Models, Molecular , Molecular Sequence Data , Peroxidase/chemistry , Peroxidase/genetics , Peroxidases/chemistry , Peroxidases/genetics , Peroxidases/metabolism , Protein Conformation , Sequence Homology, Amino Acid , Static Electricity , Nicotiana/geneticsABSTRACT
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.