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 (CA[Mn]), which shows peroxidase activity with a bicarbonate-dependent mechanism. In the presence of bicarbonate and hydrogen peroxide, (CA[Mn]) 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). CA[Mn] 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 CA[Mn] avoids the formation of aldehyde side products. CA[Mn] 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.
Okrasa
Krzysztof
K
University of Minnesota, Department of Biochemistry, Molecular Biology and Biophysics, and The Biotechnology Institute, 1479 Gortner Avenue, Saint Paul, MN 55108, USA.
Kazlauskas
Romas J
RJ