Sato Hideaki, Hayashi Takashi, Ando Tsutomu, Hisaeda Yoshio, Ueno Takafumi, Watanabe Yoshihito
PRESTO, Japan Science and Technology Agency, Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu University, Fukuoka 812-8581, Japan.
J Am Chem Soc. 2004 Jan 21;126(2):436-7. doi: 10.1021/ja038798k.
To modulate the physiological function of a hemoprotein, most approaches have been demonstrated by site-directed mutagenesis. Replacement of the native heme with an artificial prosthetic group is another way to modify a hemoprotein. However, an alternate method, mutation or heme reconstitution, does not always demonstrate sufficient improvement compared with the native heme enzyme. In the present study, to convert a simple oxygen storage hemoprotein, myoglobin, into an active peroxidase, we applied both methods at the same time. The native heme of myoglobin was replaced with a chemically modified heme 2 having two aromatic rings at the heme-propionate termini. The constructed myoglobins were examined for 2-methoxyphenol (guaiacol) oxidation in the presence of H2O2. Compared with native myoglobin, rMb(H64D.2) showed a 430-fold higher kcat/Km value, which is significantly higher than that of cytochrome c peroxidase and only 3-fold less than that of horseradish peroxidase. In addition, myoglobin-catalyzed degradation of bisphenol A was examined by HPLC analysis. The rMb(H64D.2) showed drastic acceleration (>35-fold) of bisphenol A degradation compared with the native myoglobin. In this system, a highly oxidized heme reactive species is smoothly generated and a substrate is effectively bound in the heme pocket, while native myoglobin only reversibly binds dioxygen. The present results indicate that the combination of a modified-heme reconstitution and an amino acid mutation should offer interesting perspectives toward developing a useful biomolecule catalyst from a hemoprotein.
为了调节血红蛋白的生理功能，大多数方法已通过定点诱变得到证实。用人工辅基取代天然血红素是修饰血红蛋白的另一种方法。然而，与天然血红素酶相比，另一种方法，即突变或血红素重构，并不总能显示出足够的改善。在本研究中，为了将一种简单的氧储存血红蛋白——肌红蛋白转化为活性过氧化物酶，我们同时应用了这两种方法。肌红蛋白的天然血红素被一种在血红素丙酸酯末端带有两个芳香环的化学修饰血红素2所取代。在过氧化氢存在的情况下，对构建的肌红蛋白进行了2-甲氧基苯酚（愈创木酚）氧化实验。与天然肌红蛋白相比，rMb(H64D.2)的kcat/Km值高出430倍，这显著高于细胞色素c过氧化物酶，仅比辣根过氧化物酶低3倍。此外，通过高效液相色谱分析检测了肌红蛋白催化的双酚A降解。与天然肌红蛋白相比，rMb(H64D.2)显示出双酚A降解的急剧加速（>35倍）。在这个系统中，一种高度氧化且具有反应活性的血红素物种能够顺利生成，底物能有效地结合在血红素口袋中，而天然肌红蛋白只能可逆地结合双氧。目前的结果表明，修饰血红素重构和氨基酸突变的结合应为从血红蛋白开发有用的生物分子催化剂提供有趣的前景。
