Ueyama H, Chiba Y, Kobayashi M
National Food Research Institute, Tsukuba, Japan.
Biosci Biotechnol Biochem. 1995 May;59(5):864-8. doi: 10.1271/bbb.59.864.
A fluorescent reagent, o-phthalaldehyde (OPA), competitively inhibited porcine pancreatic alpha-amylase (PPA) with Ki values of 0.7-0.9 mM, while alpha-amylase from Bacillus subtilis (BS) was uncompetitively inhibited, with Ki values of 5.8-7.6 mM. In both cases, OPA gave a time-dependent irreversible inactivation, where the amylase activity was lost faster than the maltosidase activity. Zymograms of the course of OPA modification showed that PPA was converted into at least six, faster moving components and BS gave two components. The OPA modification was retarded by the addition of the substrate analog, cyclodextrins, and the OPA modified enzymes decreased in affinity for the substrate soluble starch. Stoichiometric measurement showed that both PPA and BS was inactivated by the incorporation of 1 mol of OPA per mol of enzyme. The role of OPA modification of alpha-amylases was discussed in relation to the regulation of catalytic activity of enzymes.
荧光试剂邻苯二甲醛（OPA）对猪胰α-淀粉酶（PPA）具有竞争性抑制作用，其抑制常数（Ki）值为0.7 - 0.9 mM，而枯草芽孢杆菌（BS）的α-淀粉酶则受到非竞争性抑制，Ki值为5.8 - 7.6 mM。在这两种情况下，OPA都会导致时间依赖性的不可逆失活，其中淀粉酶活性的丧失速度比麦芽糖酶活性更快。OPA修饰过程的酶谱显示，PPA转化为至少六个迁移速度更快的组分，而BS产生两个组分。添加底物类似物环糊精会抑制OPA修饰，并且OPA修饰后的酶对底物可溶性淀粉的亲和力降低。化学计量测定表明，每摩尔酶掺入1摩尔OPA会使PPA和BS都失活。结合酶催化活性的调节讨论了OPA对α-淀粉酶修饰的作用。
