Kinetics Governing the Inhibitory Effect of 3-hydroxy-4-methoxycinnamic Acid on Tyrosinase-catalyzed Reactions
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Abstract:
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-NaH2PO4 buffer system (pH = 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.
