Histidine at the active site of Neurospora tyrosinase

Abstract

The involvement of histidyl residues as potential ligands to the binuclear active-site copper of Neurospora tyrosinase was explored by dye-sensitized photooxidation. The enzymatic activity of the holoenzyme was shown to be unaffected by exposure to light in the presence of methylene blue; however, irradiation of the apoenzyme under the same conditions led to a progressive loss of its ability to be reactivated with Cu2+. This photoinactivation was paralleled by a decrease in the histidine content whereas the number of histidyl residues in the holoenzyme remained constant. Copper measurements of photooxidized, reconstituted apoenzyme demonstrated the loss of binding of one copper atom per mole of enzyme as a consequence of photosensitized oxidation of three out of nine histidine residues. Their sequence positions were determined by a comparison of the relative yields of the histidine containing peptides of photooxidized holo- and apotyrosinases. The data obtained show the preferential modification of histidyl residues 188, 193, and 289 and suggest that they constitute metal ligands to one of the two active-site copper atoms. Substitution of copper by cobalt was found to afford complete protection of the histidyl residues from being modified by dye-sensitized photooxidation.

Ability of Various Chemicals to Reduce Copper and to Inactivate Mushroom Tyrosinase

“…In analogy with the studies of , of Pfiffener and Lerch (1981) and of Ljones and Skotland (1984) mentioned above, an alternative possibility is that the inactivation of mushroom tyrosinase by some of the reducing agents tested may proceed via the formation of free radicals close to the active site of the enzyme and that these free radicals attack an adjacent amino acid(s), probably histidine, followed by the oxidation of the latter to yield an inactivated enzyme.…”

“…According to studies of and of Pfiffener and Lerch (1981), reaction inactivation (self-inactivation during the conversion of substrate to product) and dye-sensitized photo-oxidation of tyrosinase are accompanied by a loss of binding of one of the two active site copper ions. Histidines are the ligands for the Cu2+ at the active site of the enzyme .…”

Heterologous expression and characterization of functional mushroom tyrosinase (AbPPO4)

“…Tyrosinases catalyze the ortho -hydroxylation of monophenols to o -diphenols (monophenolase or cresolase activity, EC 1.14.18.1) as well as the subsequent two-electron oxidation to the respective o -quinones (diphenolase or catechol oxidase activity, EC 1.10.3.1), which is coupled with the reduction of molecular oxygen to water 3, 4 . The active site of tyrosinase is composed of two copper ions which are coordinated by three histidine side chains each 5 forming a type III copper center 6 . Activation of molecular oxygen is affected by binding of dioxygen to the type III copper center in a characteristic ‘side-on’ bridging mode (µ-η 2 :η 2 ) 7–9 .…”

Synergism Exerted by 4‐Methyl Catechol, Catechol, and Their Respective Quinones on the Rate of DL‐DOPA Oxidation by Mushroom Tyrosinase

“…Then, 2 ml aliquots of the yellow solution obtained were withindicates that mushroom tyrosinase, originally present in the reaction mixture, was inactivated by 4-methyl-o-benzoquinone that had been formed during the first 30 min incubation of the enzyme with 4methyl catechol. Since tyrosinase is known to undergo irreversible inactivation during the oxidation of o-dihydroxyphenols to o-quinones (Golan-Goldhirsh and Whitaker, 1985;Pfiffner and Lerch, 1981; Wood and Ingraham, I%), the observation that there was an increase in absorbancy at 475 nm immediately after the addition of DGDOPA (Fig. 5B) indicates that DGDOPA was oxidized by 4-methyl-o-benzoquinone that had been formed during the first 30 min of incubation of 4-methyl catechol with mushroom tyrosinase.…”