Espín J C, García-Ruiz P A, Tudela J, García-Cánovas F
GENZ: Grupo investigación Enzimología, Departamento de Bioquímica y Biología Molecular-A, Facultad de Biología, Universidad de Murcia, A. Correos 4021, E-30080 Espinardo, Murcia, Spain.
Biochem J. 1998 Apr 15;331 ( Pt 2)(Pt 2):547-51. doi: 10.1042/bj3310547.
This paper reports experiments on the stereospecificity observed in the monophenolase and diphenolase activities of mushroom tyrosinase. Several enantiomorphs of monophenols and o-diphenols were assayed: L-tyrosine, D,L-tyrosine, D-tyrosine; L-alpha-methyltyrosine, D,L-alpha-methyltyrosine; L-dopa, D,L-dopa, D-dopa; L-alpha-methyldopa, D,L-alpha-methyldopa; L-isoprenaline, D,L-isoprenaline and D-isoprenaline. The Vmax values obtained for each series were the same. The electronic densities on the carbon atoms in the meta (C-3) and the para (C-4) positions of the benzene ring were determined by NMR assays. This value is related to the nucleophilic power of the oxygen atom belonging to the hydroxy group, which could explain the Vmax values experimentally obtained for the monophenolase and diphenolase activities of mushroom tyrosinase. The spatial orientation of the ring substituents led to lower Km values for L-isomers than for D-isomers. However, the Vmax values were the same for each series of isomers because spatial orientation did not affect the NMR value of C-4. Therefore mushroom tyrosinase showed stereospecificity in its affinity towards its substrates (Km) but not in the transformation reaction rate (Vmax) of these substrates.
本文报道了关于蘑菇酪氨酸酶单酚酶和二酚酶活性中观察到的立体特异性的实验。对几种单酚和邻二酚的对映体进行了测定：L-酪氨酸、D,L-酪氨酸、D-酪氨酸；L-α-甲基酪氨酸、D,L-α-甲基酪氨酸；L-多巴、D,L-多巴、D-多巴；L-α-甲基多巴、D,L-α-甲基多巴；L-异丙肾上腺素、D,L-异丙肾上腺素和D-异丙肾上腺素。每个系列获得的Vmax值相同。通过核磁共振测定法确定苯环间位（C-3）和对位（C-4）碳原子上的电子密度。该值与属于羟基的氧原子的亲核能力有关，这可以解释实验中获得的蘑菇酪氨酸酶单酚酶和二酚酶活性的Vmax值。环取代基的空间取向导致L-异构体的Km值低于D-异构体。然而，由于空间取向不影响C-4的核磁共振值，每个异构体系列的Vmax值相同。因此，蘑菇酪氨酸酶对其底物的亲和力（Km）表现出立体特异性，但对这些底物的转化反应速率（Vmax）没有立体特异性。
