Rodríguez-López J N, Fenoll L G, García-Ruiz P A, Varón R, Tudela J, Thorneley R N, García-Cánovas F
Grupo de Investigación de Enzimología, Departamento de Bioquímica y Biología Molecular A, Facultad de Biología, Departamento de Química Orgánica, Facultad de Química, Universidad de Murcia, E-30100 Espinardo, Murcia, Spain.
Biochemistry. 2000 Aug 29;39(34):10497-506. doi: 10.1021/bi000539+.
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.
为了阐明限速步骤并深入了解这些底物的氧化机制，通过稳态和瞬态动力学研究了蘑菇（双孢蘑菇）酪氨酸酶在几种邻二酚底物存在下与双加氧的反应。动力学分析首次确定了构成催化循环的几个部分反应的各个速率常数。蘑菇酪氨酸酶与双加氧快速反应，二级速率常数k(+8) = 2.3×10(7) M(-)(1) s(-)(1)，这与血蓝蛋白报道的速率常数[(1.3×10(6))-(5.7×10(7)) M(-)(1) s(-)(1)]相似。脱氧酪氨酸酶在双核Cu(I)位点与双加氧可逆结合，解离常数K(D)(O)()2 = 46.6 microM，这与报道的双加氧与普通章鱼脱氧血蓝蛋白结合的值（K(D)(O)()2 = 90 microM）相似[萨尔瓦托等人（1998年）《生物化学》37卷，14065 - 14077页]。瞬态和稳态动力学表明，4 - 叔丁基邻苯二酚等邻二酚与变酪氨酸酶反应（k(+2) = 9.02×10(6) M(-)(1) s(-)(1)）比与氧合酪氨酸酶反应（k(+6) = 5.4×10(5) M(-)(1) s(-)(1)）快得多。这种差异可以从调节邻二酚进入这两种酶形式活性位点的空间和极性差异效应来解释。几种邻二酚的k(cat)值也与控制底物在酪氨酸酶活性位点的迁移率、取向以及由此产生的反应性的空间和极性因素一致。
