Tokai Shota, Bito Tomohiro, Shimizu Katsuhiko, Arima Jiro
The United Graduate School of Agricultural Sciences, Tottori University, 4-101 Koyama-minami, Tottori 680-8553, Japan.
Department of Agricultural, Biological, and Environmental Sciences, Faculty of Agriculture, Tottori University, 4-101 Koyama-minami, Tottori 680-8553, Japan.
Biochem Biophys Res Commun. 2017 May 27;487(2):356-361. doi: 10.1016/j.bbrc.2017.04.064. Epub 2017 Apr 13.
Enzymes belonging to the S9 family of prolyl oligopeptidases are of interest because of their pharmacological importance and have a non-catalytic β-propeller domain. In this study, we found that the oxidation of Met203, which lies on surface of the β-propeller domain, leads to change in the substrate specificity of eryngase, an enzyme from Pleurotus eryngii and a member of the S9 family of prolyl oligopeptidases. The activity of eryngase for L-Phe-p-nitroanilide was maintained following hydrogen peroxide treatment but was dramatically reduced for other p-nitroanilide substrates. MALDI-TOF MS analysis using tryptic peptides of eryngase indicated that the change in substrate specificity was triggered by oxidizing Met203 to methionine sulfoxide. In addition, mutations of Met203 to smaller residues provided specificities similar to those observed following oxidation of the wild-type enzyme. Substitution of Met203 with Phe significantly decreased activity, indicating that Met203 may be involved in substrate gating.
由于脯氨酰寡肽酶S9家族的酶具有药理学重要性，因而备受关注，且它们具有一个非催化性的β-螺旋桨结构域。在本研究中，我们发现位于β-螺旋桨结构域表面的Met203发生氧化，会导致杏鲍菇脯氨酰寡肽酶（eryngase，属于脯氨酰寡肽酶S9家族的一种酶）的底物特异性发生变化。用过氧化氢处理后，eryngase对L-苯丙氨酸对硝基苯胺的活性得以维持，但对其他对硝基苯胺底物的活性则显著降低。使用eryngase的胰蛋白酶肽段进行的基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）分析表明，底物特异性的变化是由Met203氧化为甲硫氨酸亚砜引发的。此外，将Met203突变为较小的残基，其特异性与野生型酶氧化后观察到的特异性相似。用苯丙氨酸取代Met203会显著降低活性，这表明Met203可能参与底物门控。
