Xiao R, Solovyov A, Gilbert H F, Holmgren A, Lundström-Ljung J
Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden.
J Biol Chem. 2001 Jul 27;276(30):27975-80. doi: 10.1074/jbc.M104203200. Epub 2001 May 24.
Protein-disulfide isomerase (PDI) has five domains: a, b, b', a' and c, all of which except c have a thioredoxin fold. A single catalytic domain (a or a') is effective in catalyzing oxidation of a reduced protein but not isomerization of disulfides (Darby, N. J., and Creighton, T. E. (1995) Biochemistry 34, 11725-11735). To examine the structural basis for this oxidase and isomerase activity of PDI, shuffled domain mutants were generated using a method that should be generally applicable to multidomain proteins. Domains a and a' along with constructs ab, aa', aba', ab'a' display low disulfide isomerase activity, but all show significant reactivity with mammalian thioredoxin reductase, suggesting that the structure is not seriously compromised. The only domain order that retains significant isomerase activity has the b' domain coupled to the N terminus of the a' domain. This b'a'c has 38% of the isomerase activity of wild-type PDI, equivalent to the activity of full-length PDI with one of the active sites inactivated by mutation (Walker, K. W., Lyles, M. M., and Gilbert, H. F. (1996) Biochemistry 35, 1972-1980). Individual a and a' domains, despite their very low isomerase activities in vitro, support wild-type growth of a pdi1Delta Saccharomyces cerevisiae strain yeast. Thus, most of the PDI structure is dispensable for its essential function in yeast, and high-level isomerase activity appears not required for viability or rapid growth.
蛋白质二硫键异构酶（PDI）有五个结构域：a、b、b'、a'和c，除c外，其他结构域均具有硫氧还蛋白折叠结构。单个催化结构域（a或a'）可有效催化还原型蛋白质的氧化，但不能催化二硫键的异构化反应（达比，N. J.，和克里顿，T. E.（1995年）《生物化学》34卷，11725 - 11735页）。为了研究PDI这种氧化酶和异构酶活性的结构基础，使用了一种通常适用于多结构域蛋白质的方法构建了结构域改组突变体。结构域a和a'以及构建体ab、aa'、aba'、ab'a'显示出较低的二硫键异构酶活性，但它们与哺乳动物硫氧还蛋白还原酶均表现出显著的反应活性，这表明其结构并未受到严重破坏。唯一保留显著异构酶活性的结构域排列方式是b'结构域与a'结构域的N端相连。这种b'a'c结构的异构酶活性是野生型PDI的38%，等同于其中一个活性位点因突变而失活的全长PDI的活性（沃克，K. W.，莱尔斯，M. M.，和吉尔伯特，H. F.（1996年）《生物化学》35卷，1972 - 1980页）。尽管单个a和a'结构域在体外的异构酶活性非常低，但它们能支持pdi1Delta酿酒酵母菌株酵母的野生型生长。因此，对于其在酵母中的基本功能而言，PDI的大部分结构是可有可无的，并且对于生存能力或快速生长而言，似乎并不需要高水平的异构酶活性。
