Espín J C, Varón R, Fenoll L G, Gilabert M A, García-Ruíz P A, Tudela J, García-Cánovas F
Grupo investigación Enzimología, Departamento de Bioquímica y Biología Molecular-A, Facultad de Biología, Universidad de Murcia, Spain.
Eur J Biochem. 2000 Mar;267(5):1270-9. doi: 10.1046/j.1432-1327.2000.01013.x.
This paper reports a quantitative study of the effect of ring substituents in the 1-position of the aromatic ring on the rate of monophenol hydroxylation and o-diphenol oxidation catalyzed by tyrosinase. A possible correlation between the electron density of the carbon atom supporting the oxygen from the monophenolic hydroxyl group and the V Mmax values for each monophenol was found. In the case of o-diphenols the same effect was observed but the size of the side-chain became very important. NMR studies on the monophenols justified the sequence of the V Mmax values obtained. As regards the o-diphenols, on the other hand, only a fair correlation between NMR and V Dmax values was observed due to the effect of the molecular size of the ring substituent. From these data, it can be concluded that the redox step (k33) is not the rate-determining step of the reaction mechanism. Thus, the monophenols are converted into diphenols, but the order of specificities towards monophenols is different to that of o-diphenols. The rate-limiting step of the monophenolase activity could be the nucleophilic attack (k51) of the oxygen atom of the hydroxyl group on the copper atoms of the active site of the enzyme. This step could also be similar to or have a lower rate of attack than the electrophilic attack (k52) of the oxygen atom of the active site of oxytyrosinase on the C-3 of the monophenolic ring. However, the rate-limiting step in the diphenolase activity of tyrosinase could be related to both the nucleophilic power of the oxygen atom belonging to the hydroxyl group at the carbon atom in the 3-position (k32) and to the size of the substituent side-chain. On the basis of the results obtained, kinetic and structural models describing the monophenolase and diphenolase reaction mechanisms for tyrosinase are proposed.
本文报道了一项关于芳环1位上的环取代基对酪氨酸酶催化的单酚羟基化速率和邻二酚氧化速率影响的定量研究。发现了支持单酚羟基氧原子的碳原子的电子密度与每种单酚的V Mmax值之间可能存在的相关性。对于邻二酚，观察到了相同的效应，但侧链大小变得非常重要。对单酚的核磁共振研究证明了所获得的V Mmax值的顺序是合理的。另一方面，对于邻二酚，由于环取代基分子大小的影响，仅观察到核磁共振与V Dmax值之间有一定的相关性。从这些数据可以得出结论，氧化还原步骤（k33）不是反应机制的速率决定步骤。因此，单酚被转化为二酚，但对单酚的特异性顺序与邻二酚不同。单酚酶活性的限速步骤可能是羟基氧原子对酶活性位点铜原子的亲核攻击（k51）。这一步骤也可能与氧酪氨酸酶活性位点的氧原子对单酚环C-3的亲电攻击（k52）相似或具有更低的攻击速率。然而，酪氨酸酶的二酚酶活性的限速步骤可能与3位碳原子上羟基所属氧原子的亲核能力（k32）以及取代基侧链的大小有关。基于所获得的结果，提出了描述酪氨酸酶单酚酶和二酚酶反应机制的动力学和结构模型。
