Kinetic characterization of active site mutants Ser402Ala and Phe397His of vanadium chloroperoxidase from the fungus Curvularia inaequalis

Site-directed mutagenesis was performed on two conserved active site residues of vanadium chloroperoxidase (VCPO) from the fungus Curvularia inaequalis. Mutation of an active site residue Ser402 to Ala (S402A), a residue proposed to be brominated during turnover, caused a decrease in its activity, however it still catalyses efficiently the oxidation of both chloride and bromide. The Km values for chloride and bromide of S402A at the optimal pH 4.5 were 3.2 mM and 20 muM, respectively. The active site residues officinalis show very high VCPO and vanadium bromoperoxidases (VBPO) from the seaweeds Ascophyllum nodosum and Corallina of similarity. A prominent difference in the active site architecture of VCPO and VBPO is the presence of a second histidine in VBPO, a residue substituted by a phenylalanine in VCPO. The mutation of Phe397 to His (F397H) resulted in the enhancement of bromination activity under certain conditions. However, inactivation of F397H by halide especially at low pH was observed during turnover. Kinetic parameters and characteristics of these mutants are discussed in this report. A detailed kinetic analysis of the pH dependence of the chlorinating and brominating activity of VCPO and the S402A yielded an interesting difference between the two activities. The results show that the K-m for Cl- was pH dependent whereas the K-m for Br- was hardly pH dependent. The data confirm that protonation of an active site residue or the bound peroxide is essential for chloride oxidation. (C) 2003 Elsevier B.V. All rights reserved.