Kinetic characterization of the substrate specificity and mechanism of mushroom tyrosinase
Kinetic characterization of the substrate specificity and mechanism of mushroom tyrosinase
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DOI：
10.1046/j.1432-1327.2000.01013.x
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发表时间：
2000-03-01
期刊：
影响因子：
--
通讯作者：
García-Cánovas, F
中科院分区：
文献类型：
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作者：
Espín, JC;Varón, R;García-Cánovas, F
本文定量研究了芳香环1-位上取代基对酪氨酸酶催化单酚羟基化和邻苯二酚氧化反应速率的影响。在单酚羟基上支撑氧的碳原子的电子密度与各单酚的V-max（M）值之间可能存在相关性，在邻苯二酚中也观察到同样的效应，但侧链的大小变得非常重要。对单酚的NMR研究证明了所获得的V-max（M）值的顺序。另一方面，对于邻二酚，由于环取代基的分子大小的影响，仅观察到NMR和Vm（ax）（D）值之间的良好相关性。从这些数据可以得出结论，氧化还原步骤（k（33））不是反应机理的速率决定步骤。因此，一元酚转化为二酚，但对一元酚的特异性顺序与邻二酚的特异性顺序不同。单酚酶活性的限速步骤可能是羟基的氧原子对酶活性中心的铜原子的亲核攻击（k（51））。该步骤也可以类似于单酚环的C-3上的氧基酪氨酸酶活性位点的氧原子的亲电攻击（k（52））或具有比其更低的攻击速率。然而，酪氨酸酶的二酚酶活性中的限速步骤可能与属于3位碳原子处的羟基的氧原子的亲核能力（k（32））和取代基侧链的大小有关。在此基础上，提出了酪氨酸酶单酚酶和二酚酶反应机理的动力学和结构模型。
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-max(M) 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-max(M) values obtained. As regards the o-diphenols, on the other hand, only a fair correlation between NMR and Vm(ax)(D) 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 (k(33)) 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 (k(51)) 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 (k(52)) 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 (k(32)) 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.