Characterization and reaction mechanism of tyrosinase from neurospora crassa

Abstract

Tyrosinase (monophenol, dihydroxyphenilalanine: oxygen oxidoreductase, E. C. No. 1. 14. 18. 1) is a copper containing mixed-function oxygenase catalyzing the o-hydroxylation of monophenols and the oxidation of o-diphenols to o-quinones. The enzyme was induced by addition of cyclohexiimide in $\mbox{\underline{N}}$. $\mbox{\underline{crassa}}$ cultures. The enzyme was purified about 500-fold by following steps, that is, 60% ammonium sulfate fractionation, Celite and DEAE-Sephadex (A-50) chromatography to give a specific activity of 260.4 units per mg protein and an overall recovery of 28%. The enzyme seems to attack various monophenols and diphenols and the substituent groups on phenols appear to affect profoundly the binding and the reactivity of the enzyme. The kinetic constants for these compounds were calculated to be about $10^{-3}-10^{-4}$ M. Among the tested substrate analogues, 4-tert-butylcatechol was found to be the best substrate. From the studies on the effect of varing pH and temperature, the activity of the enzyme was found to be optimum at pH 7.0 and 40 ℃. The cresolase activity of the enzyme showed rather long (3-4 min.) lag period, which was disappeared upon the addition of trace amount of DL-DOPA ($1 × 10^{-5}$ M). Other reductants, such as ascorbate, ferrocyanide, reduced PMS and cytochrome c, had no effect on the lag. In the initial rate experiments, a series of convergent lines was obtained in double reciprocal plots, which suggests the formation of ternary complex in the enzyme action. The enzyme was severely inhibited by copper chelating agents, such as DDC or cyanide, and by thiols, like 2-mercaptoethanol. But, SH-blocking agents did not inhibit the enzyme activity. The kinetic patterns of inhibition by PABA and cyanide in both activities provided some indications that there are two binding sites for phenolic substrates. And, from the experiment of photooxidation of holo- and apoenzyme, it can be suggested that histidine may involve ...