Two Potent Suicide Substrates of Mushroom Tyrosinase: 7,8,4‘-Trihydroxyisoflavone and 5,7,8,4‘-Tetrahydroxyisoflavone

Abstract

The inhibitory characteristics of two isoflavone metabolites, 7,8,4'-trihydroxyisoflavone and 5,7,8,4'-tetrahydroxyisoflavone, on mushroom tyrosinase were investigated. The two isoflavones were isolated from soygerm koji and inhibited both monophenolase and diphenolase activities of tyrosinase. Their inhibition type was demonstrated to be irreversible inhibition by preincubation and recovery experiments. By using HPLC analysis, it was found that mushroom tyrosinase could catalyze the two isoflavones. These results revealed that the two isoflavones belonged to suicide substrates of mushroom tyrosinase. The partition ratios between molecules of suicide substrate in the formation of product and in the inactivation of enzyme were determined to be 81.7 +/- 5.9 and 35.5 +/- 3.8 for 7,8,4'-trihydroxyisoflavone and 5,7,8,4'-tetrahydroxyisoflavone, respectively. From kinetic studies, maximal inactivation rate constants and Michaelis constants were 0.79 +/- 0.08 and 1.01 +/- 0.04 min(-1) and 18.7 +/- 2.31 and 7.81 +/- 0.05 microM for 7,8,4'-trihydroxyisoflavone and 5,7,8,4'-tetrahydroxyisoflavone, respectively, when L-DOPA was used as the enzyme substrate. Structure analysis comparing the inactivating activity between the two isoflavones and their structure analogues showed that not only the 7,8-dihydroxyl groups but also the isoflavone skeleton of the two isoflavones played an important role in inactivating tyrosinase activity. The present study demonstrated that 7,8,4'-trihydroxyisoflavone and 5,7,8,4'-tetrahydroxyisoflavone are potent suicide substrates of mushroom tyrosinase.