3-羟基-4-甲氧基肉桂酸抑制酪氨酸酶催化反应的动力学研究

Kinetics Governing the Inhibitory Effect of 3-hydroxy-4-methoxycinnamic Acid on Tyrosinase-catalyzed Reactions

在30℃,p H=6.8的Na2HPO4-Na H2PO4缓冲体系中,采用酶动力学方法研究了3-羟基-4-甲氧基肉桂酸对酪氨酸酶单酚酶和二酚酶活力的影响和抑制动力学。实验结果表明,3-羟基-4-甲氧基肉桂酸对酪氨酸酶单酚酶和二酚酶活性均有良好的抑制作用。对单酚酶和二酚酶活力的相对抑制率达到50%的3-羟基-4-甲氧基肉桂酸浓度(IC50)约分别为0.13 mmol/L和0.39 mmol/L,比熊果苷抑制二酚酶活性的IC50值5.3 mmol/L小得多。3-羟基-4-甲氧基肉桂酸能明显延长单酚酶的迟滞时间,0.2 mmol/L3-羟基-4-甲氧基肉桂酸能使迟滞时间由1.1 min延长至4.3 min。对二酚酶的抑制作用表现为可逆效应,说明其是通过抑制酶活力而导致催化效率的降低,而不是通过减少有效的酶量导致酶活力的下降。Lineweaver-Burk图显示3-羟基-4-甲氧基肉桂酸对二酚酶的抑制作用表现为竞争性抑制,最大反应速率(vm)为64.5μmol/min,抑制常数(KI)为0.11 mmol/L。

The kinetics governing the inhibitory effect of 3-hydroxy-4-methoxycinnamic acid on the activities of monophenolase and diphenolase in tyrosinase were studied using an enzymological kinetic analysis method, in a Na2HPO4-Na H2PO4 buffer system(p H = 6.8) at 30 ℃. The 3-hydroxy-4-methoxycinnamic acid was observed to efficiently inhibit tyrosinase monophenolase and diphenolase activities. Approximately 0.13 mmol/L and 0.39 mmol/L 3-hydroxy-4-methoxycinnamic acid caused a 50% rate inhibition(IC50) in monophenolase and diphenolase activities, respectively; these values were much lower than that of arbutin(IC50 = 5.3 mmol/L for diphenolase activity). The presence of 3-hydroxy-4-methoxycinnamic acid also prolonged the lag period of monophenolase; the presence of 0.20 mmol/L 3-hydroxy-4-methoxycinnamic acid resulted in a lag period of 4.3 min, compared to the 1.1 min lag period of monophenolase in the absence of 3-hydroxy-4-methoxycinnamic acid. The inhibition of diphenolase by 3-hydroxy-4-methoxycinnamic acid appeared to be reversible. This indicated that the inhibitor suppresses the enzyme activity, resulting in a reduction in catalysis efficiency, as opposed to removing the effective enzyme, which leads to a reduction in enzyme activity. The Lineweaver-Burk plot demonstrated the competitive inhibition of tyrosinase by 3-hydroxy-4-methoxycinnamic acid, with a maximum reaction rate(vm) and inhibition constant(KI) of 64.5 μmol/min and 0.11 mmol/L, respectively.