Vuori K, Myllylä R, Pihlajaniemi T, Kivirikko K I
Collagen Research Unit, University of Oulu, Finland.
J Biol Chem. 1992 Apr 15;267(11):7211-4.
Protein disulfide isomerase (PDI, EC 5.3.4.1) is a highly unusual multifunctional polypeptide, being identical to the beta subunit of prolyl 4-hydroxylase, a cellular thyroid hormone binding protein and a component of the microsomal triglyceride transfer protein complex, and highly similar to a polypeptide acting in vitro as a glycosylation site binding protein. It has two -Cys-Gly-His-Cys- sequences which, it has been proposed, act as catalytic sites for the isomerase activity, but few data have been available to indicate whether one or both of them do indeed act as catalytic sites and whether the two presumed catalytic sites act independently or cooperatively. We report here on the expression of human PDI in Escherichia coli with three different signal sequences. All three polypeptide variants were secreted into the periplasmic space as fully active enzymes. Oligonucleotide-directed mutagenesis was used to convert either one or both of the -Cys-Gly-His-Cys- sequences to -Ser-Gly-His-Cys-. The PDI activity of both polypeptides containing a single modified sequence was about 50% of that of the wild-type polypeptide, whereas the polypeptide with two modified sequences had no isomerase activity. It is thus concluded that both -Cys-Gly-His-Cys- sequences act as catalytic sites for the isomerase activity, and the two catalytic sites appear to operate independently of one another.
蛋白质二硫键异构酶（PDI，EC 5.3.4.1）是一种极为特殊的多功能多肽，它与脯氨酰4 - 羟化酶的β亚基相同，是一种细胞甲状腺激素结合蛋白以及微粒体甘油三酯转运蛋白复合物的一个组分，并且与一种在体外作为糖基化位点结合蛋白起作用的多肽高度相似。它有两个-Cys-Gly-His-Cys-序列，有人提出这两个序列作为异构酶活性的催化位点，但几乎没有数据表明它们其中之一或两者是否确实作为催化位点起作用，以及这两个假定的催化位点是独立作用还是协同作用。我们在此报道了用三种不同信号序列在大肠杆菌中表达人PDI。所有三种多肽变体均作为完全有活性的酶分泌到周质空间。采用寡核苷酸定向诱变将其中一个或两个-Cys-Gly-His-Cys-序列转变为-Ser-Gly-His-Cys-。含有单一修饰序列的两种多肽的PDI活性约为野生型多肽的50%，而含有两个修饰序列的多肽则没有异构酶活性。因此可以得出结论，两个-Cys-Gly-His-Cys-序列均作为异构酶活性的催化位点，并且这两个催化位点似乎彼此独立发挥作用。
