The Isolation of Tyrosinase from Aspergillus nidulans, Its Kinetic and Molecular Properties and Some Consideration of Its Activity in vivo

SUMMARY: A single-step procedure was developed which enabled the phenol oxidase of Aspergillus nidulans to be recovered in a state of high purity. Ion-exchange chromatography was used to separate the phenol oxidase from an inhibitory protein. Kinetic analysis of the enzyme revealed that it was a tyrosinase (EC. 1.10.3.1) and had a variable ratio of o-hydroxylase to o-diphenol oxidase activity. The enzyme had a relatively low affinity for oxygen (Km of 125 μM) but, compared to other phenol oxidases, a high affinity for its phenolic substrate (Km DOPA of 250 μM). The Aspergillus tyrosinase was heterogeneous and appeared to exist in a monomer (mol. wt 1.3 × 105)-tetramer (5.2 × 105) equilibrium. The protein inhibitor was of similar size to the tyrosinase monomer and caused non-competitive inhibition of the enzyme. Both of the tyrosinase activities and the inhibitor had a substantial component of ribonucleic acid. The RNA was not removed from the enzymes or the inhibitor during purification; non-specific complexes may be formed between these proteins and RNA or true ribonucleoproteins may exist. A model is proposed for the physiological function of tyrosinase in A. nidulans under conditions where secondary metabolism does not occur.