Stopped-Flow and Steady-State Study of the Diphenolase Activity of Mushroom Tyrosinase

Abstract

The reaction of mushroom (Agaricus bisporus) tyrosinase with dioxygen in the presence of several o-diphenolic substrates has been studied by steady-state and transient-phase kinetics in order to elucidate the rate-limiting step and to provide new insights into the mechanism of oxidation of these substrates. A kinetic analysis has allowed for the first time the determination of individual rate constants for several of the partial reactions that comprise the catalytic cycle. Mushroom tyrosinase rapidly reacts with dioxygen with a second-order rate constant k(+8) = 2.3 x 10(7) M(-)(1) s(-)(1), which is similar to that reported for hemocyanins [(1.3 x 10(6))-(5.7 x 10(7)) M(-)(1) s(-)(1)]. Deoxytyrosinase binds dioxygen reversibly at the binuclear Cu(I) site with a dissociation constant K(D)(O)()2 = 46.6 microM, which is similar to the value (K(D)(O)()2 = 90 microM) reported for the binding of dioxygen to Octopus vulgaris deoxyhemocyanin [Salvato et al. (1998) Biochemistry 37, 14065-14077]. Transient and steady-state kinetics showed that o-diphenols such as 4-tert-butylcatechol react significantly faster with mettyrosinase (k(+2) = 9.02 x 10(6) M(-)(1) s(-)(1)) than with oxytyrosinase (k(+6) = 5.4 x 10(5) M(-)(1) s(-)(1)). This difference is interpreted in terms of differential steric and polar effects that modulate the access of o-diphenols to the active site for these two forms of the enzyme. The values of k(cat) for several o-diphenols are also consistent with steric and polar factors controlling the mobility, orientation, and thence the reactivity of substrates at the active site of tyrosinase.

Mechanistic Studies on the Tyrosinase‐Catalyzed Formation of the Anachelin Chromophore

“…Related studies on the tyrosinasemediated oxidation of catechols suggest this step to be ratelimiting. [29,30] Interestingly, this quinone has two electrophilic carbon atoms available for nucleophilic 1,4-addition. Addition to the C atom at the C(1) position would furnish the five-membered ring spiro compound III, a reaction that has been postulated for the biosynthesis of some natural products such as FR901483 [31,32] or TAN1251 A.…”

Inhibitory effects of Cefazolin and Cefodizime on the activity of mushroom tyrosinase

“…Tyrosinase is composed of four subunits and contains two binuclear coppers in its active sites per tetramer [22]. In the process of catalysis, tyrosinase has three existing forms, E met , E oxy and E deoxy [23].…”

Melanogenesis Inhibition Due to NADH

“…This confirms that the form that accumulates in the steady state is E ox , since the binding of substrates to E ox is slower than to E m and the oxidation constant in the complex E ox S R is lower than in E m S R . 11) In the case of the catalase activity of TYR (Fig. 6, recording b), the presence of tropolone in the same concentrations as in Fig.…”

“…Under aerobic conditions at oxygen concentrations of 0.26 mM, practically the only forms existing are E ox and E m . 11) Note that the difference between k ox i and k m i is three orders of magnitude, a difference that can be used to evaluate these enzymatic forms. The experimental method is described in reference 21.…”

“…In the steady state, the predominant form is E ox , 11) as seen from the timedependent inhibition shown by tropolone. This compound is a time-dependent inhibitor of TYR and acts on the E ox form, which is accumulated when TYR acts on TBC (Fig.…”