Purification and Characterization of a Tyrosinase from Streptomyces glaucescens

1 The purification of a tyrosinase from cell-free extracts of Streptomyces glaucescens is described. The enzyme obtained is homogeneous according to the criteria of ultracentrifuge analysis and polyacrylamide gel electrophoresis. 2 An average molecular weight of 29100 was determined by dodecylsulfate-gel electrophoresis and by sedimentation equilibrium analysis in the ultracentrifuge. The amino acid composition of the pure enzyme is also reported. 3 The enzyme contains one atom of copper in the cuprous form per molecule and is strongly inhibited by KCN and 4-nitro-catechol. 4 The enzyme shows optimal activity at pH 6.8; it has an isoelectric point at pH 6.95 and is most stable at pH 8.0. 5 Variations of Km values between pH 5.0 and 8.9 when l-tyrosine methylester and 3,4-di-hydroxy-l-phenylalanine were used as substrate was interpreted by assuming that different ionizable groups are involved in the oxidation of these two substrates. 6 The Michaelis-Menten constants and KCat are strongly dependent on the oxygen concentration for 3,4-dihydroxy-l-phenylalanine and almost independent for l-tyrosine methyl ester. Kinetic parameters obtained for a series of mono- and o-diphenol substrates are also reported.