Purification, crystallization, and characterization of peroxidase from Coprinus cinereus.
Peroxidase (donor: H2O2 oxidoreductase [EC 1.11.1.7]) was purified from a culture broth of an inkcap Basidiomycete, Coprinus cinereus S.F. Gray. A single component containing a low amount of carbohydrate was isolated by affinity chromatography on concanavalin A-Sepharose and crystallized from ammonium sulfate solution. The enzyme is an acidic protein (pI 3.5) and consists of a single polypeptide chain having the molecular weight of 41,600 daltons. The enzyme contains one protohemin per molecule and exhibits the characteristic absorption, circular dichroism, and magnetic circular dichroism spectra of a heme-protein. The Coprinus peroxidase forms two characteristic intermediate compounds, I and II, and the rate constants for hydrogen peroxide and guaiacol had similar values to those for higher plant peroxidases. The ferric enzyme formed a cyanide compound with a dissociation constant similar to those for higher plant enzyme, but the dissociation constant of the ferrous enzyme-cyanide was large. The chemical composition of Coprinus peroxidase showed 381 amino acid residues, 1 glucosamine, 3 true sugars, 3 calcium, and 1 non-heme iron other than 1 protohemin. The secondary structure of the fungal enzyme was very similar to that of horseradish peroxidase.
Morita
Y
Y
Research Institute for Food Science, Kyoto University.
Yamashita
H
H
Mikami
B
B
Iwamoto
H
H
Aibara
S
S
Terada
M
M
Minami
J
J
D000363:Agaricales / Q000201:enzymology*; D000596:Amino Acids / Q000032:analysis; D002118:Calcium / Q000032:analysis; D002241:Carbohydrates / Q000032:analysis; D002846:Chromatography, Affinity; D003302:Coprinus / Q000201:enzymology*; D003460:Crystallization; D004589:Electrophoresis, Disc; D006427:Hemin / Q000032:analysis; D007501:Iron / Q000032:analysis; D007700:Kinetics; D008970:Molecular Weight; D010544:Peroxidases / Q000302:isolation & purification*; D013053:Spectrophotometry; D014461:Ultracentrifugation
D000596:Amino Acids; D002241:Carbohydrates; D006427:Hemin; D007501:Iron; D010544:Peroxidases; D002118:Calcium