酪胺酸酶與苯肼的反應機構探討

Abstract

本研究證明酪氨酸酶(tyrosinase)可以直接與苯肼(phenylhydrazine)產生氧化反應，且反應的產物是酚(phenol)。苯肼對酪氨酸酶並不是一個天然受質，由麴酸(kojic acid)抑制酪氨酸酶對苯肼氧化反應的結果，間接證實酪氨酸酶是直接與苯肼反應。超氧分子(superoxide, O2¬¬¬-)是苯肼氧化反應的中間產物，由加入超氧化物歧化酵素(superoxide dismutase; SOD) 的實驗證實，超氧分子會抑制苯肼的氧化反應；當超氧分子除去時，酪氨酸酶與苯肼的反應速率大幅增加，推測兩種可能，其一，酪氨酸酶的氧化反應產生大量的超氧分子，導致抑制苯肼的氧化反應；其二是超氧分子分解產生雙氧水，使酪氨酸酶轉變為催化活性較大的Oxy-form酪氨酸酶。加入過氧化氫酶(catalase)的反應中，證實雙氧水(H2O2)是中間產物，但在反應初期沒有雙氧水的累積，而在反應一段時間後才會有明顯的雙氧水累積。從反應環境含有雙氧水水溶液的實驗，觀察到雙氧水對於二價銅離子的反應速率大於酪氨酸酶，雙氧水幫助二價銅離子的電子傳遞，加速苯肼的氧化反應；酪氨酸酶的反應中心，雙銅離子受蛋白質包覆，雙氧水無法有效的幫助銅離子電子傳遞的加速反應，但是雙氧水可以使酪氨酸酶變成反應活性大的含氧態(oxy-form)的酪氨酸酶，加速苯肼氧化反應速率。 乙醯苯肼與酪氨酸酶的反應，顯示其反應速率很小(相較於苯肼)，推測需要大量的酪氨酸酶與長時間的反應，才有較好的去保護效率；在鹼性環境中有利於反應，推測可能是乙醯苯肼在鹼性環境中較容易水解，產生苯肼再與酵素反應。

Direct oxidation of phenylhydrazine by tyrosinase was studied. The final product of oxidation of phenylhydrazine by tyrosinase or Cu(II) is phenol. Phenylhydrazine is not the natural substrates by tyrosinase. Kojic acid is the inhibitor with oxidation of phenylhydrazine by tyrosinase. By adding SOD (superoxide dismntase), the superoxide was removed, and the rate constants were enhanced. The evidences demonstrate that superoxide is generated in the reaction and retards the oxidation of phenylhydrazine. The removal of H2O2 by adding catalase slightly slows down the reaction. H2O2 is also an intermediate in the reaction and can facilitate the oxidation of phenylhydrazine. Addition of H2O2 in the reaction increases the rate constants. The oxidation of acetylphenyl- hydrazine by tyrosinase or Cu(II) was relatively slow, compared to that of phenylhydrazine. This is due to poor removal of proton form acetylphenylhydrazine by tyrosinase or Cu(II).