Open AccessCharacterisation of a haem active‐site mutant of horseradish peroxidase, Phe41 → Val, with altered reactivity towards hydrogen peroxide and reducing substrates
Author(s) -
SMITH Andrew T.,
SANDERS Stephen A.,
THORNELEY Roger N. F.,
BURKE Julian F.,
BRAY Robert R. C.
Publication year - 1992
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1992.tb17077.x
Subject(s) - horseradish peroxidase , chemistry , hydrogen peroxide , substrate (aquarium) , peroxidase , reaction rate constant , reactivity (psychology) , stereochemistry , steady state (chemistry) , enzyme kinetics , dissociation constant , enzyme , kinetics , active site , biochemistry , organic chemistry , medicine , oceanography , physics , alternative medicine , receptor , pathology , quantum mechanics , geology
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 ( k 1 ) revealed an eightfold decrease in the reactivity of the Phe41→Val variant towards H 2 O 2 , in comparison with HRP‐C or HRP‐C * . Measurement of the remaining rate constants, K 2 and k 3 , 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 ( k u ), assumed to be product dissociation, becomes rate‐limiting under our standard assay conditions. Calculation of rate constants from steady‐state data yielded values of k 1 for both enzyme forms in adequate agreement with those from pre‐steady state measurements. They showed, furthermore, that both k 3 for 2,2′‐azinobis(3‐ethylbenzthizzoline‐6‐sulphonate) and k u were substantially decreased, fivefold and tenfold, respectively in the mutant. Analogous to the decrease in k u , 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 H 2 O 2 , 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.