title: Mechanistic studies of the tyrosinase-catalyzed oxidative cyclocondensation of 2-aminophenol to 2-aminophenoxazin-3-one.
abstract: Tyrosinase (EC 1.14.18.1) catalyzes the monophenolase and diphenolase reaction associated with vertebrate pigmentation and fruit/vegetable browning. Tyrosinase is an oxygen-dependent, dicopper enzyme that has three states: Emet, Eoxy, and Edeoxy. The diphenolase activity can be carried out by both the met and the oxy states of the enzyme while neither mono- nor diphenolase activity results from the deoxy state. In this study, the oxidative cyclocondensation of 2-aminophenol (OAP) to the corresponding 2-aminophenoxazin-3-one (APX) by mushroom tyrosinase was investigated. Using a combination of various steady- and pre-steady state methodologies, we have investigated the kinetic and chemical mechanism of this reaction. The kcat for OAP is 75 ± 2s(-1), K(OAP)M = 1.8 ± 0.2mM, K(O2)M =25 ± 4 μM with substrates binding in a steady-state preferred fashion. Stopped flow and global analysis support a model where OAP preferentially binds to the oxy form over the met (k7 ≫ k1). For the met form, His269 and His61 are the proposed bases, while the oxy form uses the copper-peroxide and His61 for the sequential deprotonation of anilinic and phenolic hydrogens. Solvent KIEs show proton transfer to be increasingly rate limiting for kcat/K(OAP)M as [O2] → 0 μM (1.38 ± 0.06) decreasing to 0.83 ± 0.03 as [O2] → ∞ reflecting a partially rate limiting μ-OH bond cleavage (E met) and formation (E oxy) following protonation in the transition state. The coupling and cyclization reactions of o-quinone imine and OAP pass through a phenyliminocyclohexadione intermediate to APX, forming at a rate of 6.91 ± 0.03 μM(-1)s(-1) and 2.59E-2 ± 5.31E-4s(-1). Differences in reactivity attributed to the anilinic moiety of OAP with o-diphenols are discussed.
authors.affiliation: Division of Mathematical and Physical Sciences, Department of Chemistry, Xavier University of Louisiana, New Orleans, LA 70125, USA.
authors.forename: Courtney
authors.initials: C
authors.lastname: Washington
authors.affiliation: Division of Mathematical and Physical Sciences, Department of Chemistry, Xavier University of Louisiana, New Orleans, LA 70125, USA.
authors.forename: Jamere
authors.initials: J
authors.lastname: Maxwell
authors.affiliation: Division of Mathematical and Physical Sciences, Department of Chemistry, Xavier University of Louisiana, New Orleans, LA 70125, USA.
authors.forename: Joenathan
authors.initials: J
authors.lastname: Stevenson
authors.affiliation: Division of Mathematical and Physical Sciences, Department of Chemistry, Xavier University of Louisiana, New Orleans, LA 70125, USA.
authors.forename: Gregory
authors.initials: G
authors.lastname: Malone
authors.affiliation: Center for Structural Biology, Vanderbilt University, Nashville, TN 37232, USA.
authors.forename: Edward W
authors.initials: EW
authors.lastname: Lowe
authors.affiliation: Division of Mathematical and Physical Sciences, Department of Chemistry, Xavier University of Louisiana, New Orleans, LA 70125, USA.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Division of Mathematical and Physical Sciences, Department of Chemistry, Xavier University of Louisiana, New Orleans, LA 70125, USA.
authors.forename: Guangdi
authors.initials: G
authors.lastname: Wang
authors.affiliation: Division of Mathematical and Physical Sciences, Department of Chemistry, Xavier University of Louisiana, New Orleans, LA 70125, USA. Electronic address: nmcintyr@xula.edu.
authors.forename: Neil R
authors.initials: NR
authors.lastname: McIntyre
mesh_terms: D000363:Agaricales / Q000201:enzymology* / Q000378:metabolism; D000627:Aminophenols / Q000378:metabolism*; D003500:Cyclization; D007700:Kinetics; D008958:Models, Molecular; D014442:Monophenol Monooxygenase / Q000378:metabolism*; D010078:Oxazines / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism
chemical_list: D000627:Aminophenols; D010078:Oxazines; C005512:3-aminophenoxazone; C027667:2-aminophenol; D014442:Monophenol Monooxygenase; D010100:Oxygen
keywords: 2-Aminophenol; Cyclocondensation; Isotope effect; Kinetics; Mechanism; Tyrosinase