REDOX PROPERTIES AND CROSS-LINKING OF THE DITHIOL DISULFIDE ACTIVE-SITES OF MAMMALIAN PROTEIN DISULFIDE-ISOMERASE
REDOX PROPERTIES AND CROSS-LINKING OF THE DITHIOL DISULFIDE ACTIVE-SITES OF MAMMALIAN PROTEIN DISULFIDE-ISOMERASE
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DOI：
10.1042/bj2750341
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发表时间：
1991-04-15
影响因子：
4.1
通讯作者：
FREEDMAN, RB
中科院分区：
文献类型：
--
作者：
HAWKINS, HC;DENARDI, M;FREEDMAN, RB
1. PDI的活性位点二硫醇/二硫化物基团的氧化还原性质通过用过量的GSH + GSSG平衡酶，快速烷基化酶的二硫醇形式以不可逆地使其分解，并通过在标准条件下测量残余活性来确定二硫化物形式的比例来确定。2.还原的程度随所施加的氧化还原电位而变化;第一近似，数据拟合的模型中，所有的酶二硫醇/二硫化物基团是独立的和等效的，这些网站和GSH/GSSG氧化还原对之间的平衡常数是42 μ M，在pH 7.5。3.从该结果计算PDI活性位点二硫醇/二硫化物对的标准氧化还原电位，发现为-0.11V;因此PDI是比GSH、烟酰胺辅因子、硫氧还蛋白和二硫苏糖醇更强的氧化剂和更弱的还原剂。4.具有GSH/GSSG氧化还原电对的PDI的氧化还原平衡数据显示出与线性的S形偏差。假设希尔系数为1.5，可以近似地模拟S形。5. PDI二聚体中的四个活性位点之间的协同相互作用的证据通过研究PDI和反应基团之间具有各种长度的同双功能烷化剂之间的反应来扩展。一个物种的电泳迁移率表明，它包含一个链内交联观察在所有情况下，而有没有证据的PDI同源二聚体的链之间的交联。最有效的交联是用含有五个或更多个亚甲基间隔基团的试剂实现的，这意味着PDI多肽的两个硫氧还蛋白样结构域内的活性位点反应基团之间的最小距离为1.6 nm（16埃）。
1. The redox properties of the active-site dithiol/disulphide groups of PDI were determined by equilibrating the enzyme with an excess of GSH + GSSG, rapidly alkylating the dithiol form of the enzyme to inactivate it irreversibly, and determining the proportion of the disulphide form by measuring the residual activity under standard conditions. 2. The extent of reduction varied with the applied redox potential; to a first approximation, the data fitted a model in which all the enzyme dithiol/disulphide groups are independent and equivalent and the equilibrium constant between these sites and the GSH/GSSG redox couple is 42-mu-M at pH 7.5. 3. The standard redox potential for PDI active-site dithiol/disulphide couples was calculated from this result and found to be -0.11 V; hence PDI is a stronger oxidant and weaker reductant than GSH, nicotinamide cofactors, thioredoxin and dithiothreitol. 4. The redox equilibrium data for PDI with the GSH/GSSG redox couple showed sigmoidal deviations from linearity. The sigmoidicity could be modelled closely by assuming a Hill coefficient of 1.5. 5.This evidence of co-operative interactions between the four active sites in a PDI dimer was extended by studying the reaction between PDI and homobifunctional alkylating agents with various lengths between the reactive groups. A species whose electrophoretic mobility suggested it contained an intrachain cross-link was observed in all cases, whereas there was no evidence for cross-linking between the chains of the PDI homodimer. Most effective cross-linking was achieved with reagents containingfive or more methylene spacer groups, implying a minimum distance of 1.6 nm (16 angstrom) between the active-site reactive groups within the two thioredoxin-like domains of the PDI polypeptide.