Characterisation of a haem active‐site mutant of horseradish peroxidase, Phe41 → Val, with altered reactivity towards hydrogen peroxide and reducing substrates

• DOI: 10.1111/j.1432-1033.1992.tb17077.x
• Publication Date: 2005-03-04T07:06:19Z
• Authors: Andrew T. SMITH; Stephen A. SANDERS; Roger N. F. THORN...; Julian F. BURKE; Robert R. C. BRAY
• Keywords: Models, Molecular; 0301 basic medicine; Binding Sites; Base Sequence; Phenylalanine; Spectrum Analysis; Molecular Sequence Data; Valine; Heme; Hydrogen Peroxide; Recombinant Proteins; Kinetics; Structure-Activity Relationship; 03 medical and health sciences; Oligodeoxyribonucleotides; Mutation; Mutagenesis, Site-Directed; Amino Acid Sequence; Oxidation-Reduction; Horseradish Peroxidase

A horseradish peroxidase variant ([F41V] HRP‐C*), in which Val replaces the conserved Phe at position 41 adjacent to the distal His, has been constructed. Its composition and spectroscopic, catalytic and substrate‐binding properties were compared with those of the wild type recombinant (HRP‐C*) and plant (HRP‐C) enzymes. Presteady‐state kinetic measurments of the rate constant for compound I formation (k1) revealed an eightfold decrease in the reactivity of the Phe41→Val variant towards H2O2, in comparison with HRP‐C or HRP‐C*. Measurement of the remaining rate constants, K2 and k3, for the two single‐electron reduction reactions of [F41V] HRP‐C with para‐aminobenzoic acid as reducing substrate, showed that the were 2.5‐fold and 1.3‐fold faster, respectively. In contrast, analysis of data from steady‐state assays with 2,2′‐azinobis(3‐ethylbenzthiazoline‐6‐sulphonate) as reducing substrate, showed decreased reactivity of the mutant enzyme to this compound, indicating a change in substrate specificity. Over the substrate range studied, the data for HRP‐C* and for [F41V] HRP‐C conformed to a simple modification of the accepted peroxidase mechanism in which first‐order step (ku), assumed to be product dissociation, becomes rate‐limiting under our standard assay conditions. Calculation of rate constants from steady‐state data yielded values of k1 for both enzyme forms in adequate agreement with those from pre‐steady state measurements. They showed, furthermore, that both k3 for 2,2′‐azinobis(3‐ethylbenzthizzoline‐6‐sulphonate) and ku were substantially decreased, fivefold and tenfold, respectively in the mutant. Analogous to the decrease in ku, we observed a towfold increase in the affinity of the mutant variant for the inhibitor benzhydroxamic acid. The coordination‐state equilibrium of the haem iron also appeared shifted towards the hexacoordinate high‐spin form. These observations indicate that in additions to affecting reactivity to H2O2, mutations in the distal region and close to the haem iron also affect reactivity towards different reducing substrates, inducing perturbations in the neighbourhood of the aromatic‐substrate‐binding site, known to be 0.8–1.2 nm from the haem iron.