Kato Dai-ichiro, Yokoyama Keisuke, Hiraishi Yoshihiro, Takeo Masahiro, Negoro Seiji
Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo.
Biosci Biotechnol Biochem. 2011;75(9):1758-62. doi: 10.1271/bbb.110299. Epub 2011 Sep 7.
Measurement of thioesterification activities for dodecanoic acid (C12) and ketoprofen was done using five firefly luciferases, from Pyrocoelia miyako (PmL), Photinus pyralis (PpL), Luciola cruciata (LcL), Hotaria parvura (HpL), and Luciola mingrelica (LmL). Among these, PmL, PpL, and LcL showed the expected thioesterification activities toward both substrates. All the enzymes exhibited (R)-enantioselectivity toward ketoprofen, which had same tendency as firefly luciferase from Luciola lateralis (LUC-H). HpL and LmL, however, did not accept ketoprofen, although they had thioesterification activity toward C12. These results indicate that the substrate acceptance of luciferases for the thioesterification reaction varies dramatically relying on the origin of firefly. Hence we focused primarily on PmL and investigated the effect of pH on enzymatic activity. In addition, by determining the kinetic parameters at various pH values, we verified that the k(cat) parameter contributed to the preferential enantioselectivity of this enzyme.
利用来自宫古扁萤（PmL）、萤火虫（PpL）、黑翅萤（LcL）、姬红萤（HpL）和明雷萤（LmL）的五种萤火虫荧光素酶，对十二烷酸（C12）和酮洛芬的硫酯化活性进行了测定。其中，PmL、PpL和LcL对两种底物均表现出预期的硫酯化活性。所有酶对酮洛芬均表现出（R）-对映选择性，这与侧条扁萤荧光素酶（LUC-H）具有相同的趋势。然而，HpL和LmL虽然对C12具有硫酯化活性，但不接受酮洛芬。这些结果表明，荧光素酶对硫酯化反应的底物接受能力因萤火虫的来源不同而有很大差异。因此，我们主要关注PmL，并研究了pH对酶活性的影响。此外，通过测定不同pH值下的动力学参数，我们证实了k(cat)参数促成了该酶的优先对映选择性。
