Peroxidase and phosphatase activity of active-site mutants of vanadium chloroperoxidase from the fungus Curvularia inaequalis - Implications for the catalytic mechanisms

Mutation studies were performed on active site residues of vanadium chlorop eroxidase from the fungus Curvularia inaequalis, an enzyme which exhibits b oth haloperoxidase and phosphatase activity and is related to glucose-6 pho sphatase. The effects of mutation to alanine on haloperoxidase activity wer e studied for the proposed catalytic residue His-404 and for residue Asp-29 2, which is located close to the vanadate cofactor. The mutants were strong ly impaired in their ability to oxidize chloride but still oxidized bromide , although they inactivate during turnover. The effects on the optical abso rption spectrum of vanadium chloroperoxidase indicate that mutant H404A has a reduced affinity for the cofactor, whereas this affinity is unchanged in mutant D292A. The effect on the phosphatase activity of the apoenzyme was investigated for six mutants of putative catalytic residues, Effects of mut ation of His-496, Arg-490, Arg-360, Lys-353, and His-404 to alanine are in line with their proposed roles in nucleophilic attack, transition-state sta bilization, and leaving-group protonation, Asp-292 is excluded as the group that protonates the leaving group. A model based on the mutagenesis studie s is presented and may serve as a template for glucose-6-phosphatase and ot her related phosphatases. Hydrolysis of a phospho-histidine intermediate is the rate-determining step in the phosphatase activity of apochloroperoxida se, as shown by burst kinetics.