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Mechanistic studies of the tyrosinase-catalyzed oxidative cyclocondensation of 2-aminophenol to 2-aminophenoxazin-3-one

2015; Elsevier BV; Volume: 577-578; Linguagem: Inglês

10.1016/j.abb.2015.04.007

1096-0384

Courtney Washington, Jamere Maxwell, Joenathan Stevenson, G. Ray Malone, Edward W. Lowe, Qiang Zhang, Guangdi Wang, Neil McIntyre,

Synthesis and Biological Evaluation

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.