Manganese-substituted carbonic anhydrase as a new peroxidase

Carbonic anhydrase is a zinc metalloenzyme that catalyzes the hydration of carbon dioxide to bicarbonate. Replacing the active-site zinc with manganese yielded manganese-substituted carbonic anhydrase (CAMn]), which shows peroxidase activity with a bicarbonate-dependent mechanism. In the presence of bicarbonate and hydrogen peroxide, (CAMn]) catalyzed the efficient oxidation of o-dianisidine with kcat/KM=1.4 x 10(6) m(-1) s(-1), which is comparable to that for horseradish peroxidase, kcat/KM=57 x 10(6) m(-1) s(-1). CAMn] also catalyzed the moderately enantioselective epoxidation of olefins to epoxides (E=5 for p-chlorostyrene) in the presence of an amino-alcohol buffer, such as N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES). This enantioselectivity is similar to that for natural heme-based peroxidases, but has the advantage that CAMn] avoids the formation of aldehyde side products. CAMn] degrades during the epoxidation limiting the yield of the epoxidations to <12 %. Replacement of active-site residues Asn62, His64, Asn67, Gln92, or Thr200 with alanine by site-directed mutagenesis decreased the enantioselectivity demonstrating that the active site controls the enantioselectivity of the epoxidation.