对羟基苯乙酸在酪氨酸酶催化中的动力学研究

Inhibitory kinetics of p-hydroxyphenylacetic acid on mushroom tyrosinase-catalyzing reaction

采用酶动力学方法,研究了对羟基苯乙酸对蘑菇酪氨酸酶单酚酶和二酚酶酶活的动力学。实验结果表明,对羟基苯乙酸对酪氨酸酶单酚酶具有抑制作用,对单酚酶酶活的相对抑制率达到50%的对羟基苯乙酸浓度(IC50)为0.79mmol/L,并能明显延长单酚酶的延滞时间,0.8mmol/L的对羟基苯乙酸能使延滞时间由95s延长至182s。对于二酚酶活性,对羟基苯乙酸激活浓度为0.8mmol/L,其对二酚酶酶活的激活率达108%。Lineweaver-Burk图显示对羟基苯乙酸对二酚酶的激活作用表现为竞争性和反竞争性的混合型激活,对游离酶的激活常数和对酶-底物配合物的激活常数分别为-0.16和-0.19mmol/L。

The kinetic effects of p-hydroxyphenylacetic acid on the monophenolase and diphenolase activities of mushroom tyrosinase have been investigated.The results showed that p-hydroxyphenylacetic acid could inhibit the monophenolase activity and activate the diphenolase activity of mushroom tyrosinase,respectively.Moreover,p-hydroxyphenylacetic acid can also extend the lag time of catalytic oxidation of L-tyrosine by monophenolase and the marked lag period was 95s in the absence of p-hydroxyphenylacetic acid and 182 s in the presence of 0.8 mmol/L of p-hydroxyphenylacetic acid.The p-hydroxyphenylacetic acid concentration （IC50） leading to 50% activity loss was estimated to be 0.79 mmol/L for monophenolase.The activation ratio （catalytic oxidation of L-DOPA） was 108%,when the p-hydroxyphenylacetic acid concentration reached 0.8 mmol/L.Kinetic analysis showed that the activation effect of p-hydroxyphenylacetic acid on the diphenolase activity belonged to a reversible competitive-uncompetitive mixed-type one,and the equilibrium constants for activating the bonding with free enzyme and enzyme-substract （ES） were-0.16 and-0.19 mmol/L,respectively.