title: Extracellular tyrosinase from the fungus Trichoderma reesei shows product inhibition and different inhibition mechanism from the intracellular tyrosinase from Agaricus bisporus.
abstract: Tyrosinase (EC 1.14.18.1) is a widely distributed type 3 copper enzyme participating in essential biological functions. Tyrosinases are potential biotools as biosensors or protein crosslinkers. Understanding the reaction mechanism of tyrosinases is fundamental for developing tyrosinase-based applications. The reaction mechanisms of tyrosinases from Trichoderma reesei (TrT) and Agaricus bisporus (AbT) were analyzed using three diphenolic substrates: caffeic acid, L-DOPA (3,4-dihydroxy-l-phenylalanine), and catechol. With caffeic acid the oxidation rates of TrT and AbT were comparable; whereas with L-DOPA or catechol a fast decrease in the oxidation rates was observed in the TrT-catalyzed reactions only, suggesting end product inhibition of TrT. Dopachrome was the only reaction end product formed by TrT- or AbT-catalyzed oxidation of L-DOPA. We produced dopachrome by AbT-catalyzed oxidation of L-DOPA and analyzed the TrT end product (i.e. dopachrome) inhibition by oxygen consumption measurement. In the presence of 1.5mM dopachrome the oxygen consumption rate of TrT on 8mM L-DOPA was halved. The type of inhibition of potential inhibitors for TrT was studied using p-coumaric acid (monophenol) and caffeic acid (diphenol) as substrates. The strongest inhibitors were potassium cyanide for the TrT-monophenolase activity, and kojic acid for the TrT-diphenolase activity. The lag period related to the TrT-catalyzed oxidation of monophenol was prolonged by kojic acid, sodium azide and arbutin; contrary it was reduced by potassium cyanide. Furthermore, sodium azide slowed down the initial oxidation rate of TrT- and AbT-catalyzed oxidation of L-DOPA or catechol, but it also formed adducts with the reaction end products, i.e., dopachrome and o-benzoquinone.
authors.affiliation: VTT Technical Research Centre of Finland, Espoo, Finland. ext-Chiara.Gasparetti@vtt.fi
authors.forename: Chiara
authors.initials: C
authors.lastname: Gasparetti
authors.forename: Emilia
authors.initials: E
authors.lastname: Nordlund
authors.forename: Janne
authors.initials: J
authors.lastname: Jänis
authors.forename: Johanna
authors.initials: J
authors.lastname: Buchert
authors.forename: Kristiina
authors.initials: K
authors.lastname: Kruus
mesh_terms: D000364:Agaricus / Q000201:enzymology*; D002109:Caffeic Acids / Q000737:chemistry; D002396:Catechols; D003373:Coumaric Acids / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D045563:Indolequinones / Q000737:chemistry; D007424:Intracellular Fluid / Q000201:enzymology; D007700:Kinetics; D007980:Levodopa / Q000737:chemistry; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D010084:Oxidation-Reduction; D011190:Potassium Cyanide / Q000737:chemistry; D011753:Pyrones / Q000737:chemistry; D019810:Sodium Azide / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D014242:Trichoderma / Q000201:enzymology*
chemical_list: D002109:Caffeic Acids; D002396:Catechols; D003373:Coumaric Acids; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D045563:Indolequinones; D011753:Pyrones; C001123:dopachrome; D007980:Levodopa; C011890:kojic acid; D019810:Sodium Azide; D014442:Monophenol Monooxygenase; C034221:catechol; D011190:Potassium Cyanide; C040048:caffeic acid