CATALYSIS OF THE OXIDATIVE FOLDING OF RIBONUCLEASE-A BY PROTEIN DISULFIDE ISOMERASE - PRE-STEADY-STATE KINETICS AND THE UTILIZATION OF THE OXIDIZING EQUIVALENTS OF THE ISOMERASE
CATALYSIS OF THE OXIDATIVE FOLDING OF RIBONUCLEASE-A BY PROTEIN DISULFIDE ISOMERASE - PRE-STEADY-STATE KINETICS AND THE UTILIZATION OF THE OXIDIZING EQUIVALENTS OF THE ISOMERASE
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DOI：
10.1021/bi00217a005
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发表时间：
1991-01-22
期刊：
影响因子：
2.9
通讯作者：
GILBERT, HF
中科院分区：
文献类型：
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作者：
LYLES, MM;GILBERT, HF
在低浓度的谷胱甘肽氧化还原缓冲液中，蛋白质二硫键异构酶（PDI）催化的还原型核糖核酸酶A的氧化复性表现出快速但不完全的核糖核酸酶活化，这先于稳态反应。 这种行为可以归因于GSSG依赖性的底物，还原核糖核酸酶，在两类巯基/二硫化物氧化还原形式之间的分配，这些巯基/二硫化物氧化还原形式可以在低浓度的GSH下转化为活性核糖核酸酶，而那些不能。 在催化浓度的PDI和接近化学计量浓度的谷胱甘肽二硫化物的情况下，非常快速地形成约4当量（2当量的核糖核酸酶二硫化物）的GSH，随后较慢地形成GSH，这对应于另外的2个二硫键当量。 RNA酶二硫键的快速形成和随后的不正确的二硫键异构体对活性RNA酶的修复都由PDI催化。 在不存在GSSG或其他氧化剂的情况下，PDI的二硫键等价物可用于在化学计量反应中在RNA酶中形成二硫键。 在没有谷胱甘肽氧化还原缓冲液的情况下，还原核糖核酸酶再生的速率随着低于当量点的PDI浓度的增加而显著增加;然而，超过化学计量浓度的PDI抑制RNA酶再生。
At low concentrations of a glutathione redox buffer, the protein disulfide isomerase (PDI) catalyzed oxidative renaturation of reduced ribonuclease A exhibits a rapid but incomplete activation of ribonuclease, which precedes the steady-state reaction. This behavior can be attributed to a GSSG-dependent partitioning of the substrate, reduced ribonuclease, between two classes of thiol/disulfide redox forms, those that can be converted to active ribonuclease at low concentrations of GSH and those that cannot. With catalytic concentrations of PDI and near stoichiometric concentrations of glutathione disulfide, approximately 4 equiv (2 equiv of ribonuclease disulfide) of GSH are formed very rapidly followed by a slower formation of GSH, which corresponds to an additional 2 disulfide bond equiv. The rapid formation of RNase disulfide bonds and the subsequent rearrangment of incorrect disulfide isomers to active RNase are both catalyzed by PDI. In the absence of GSSG or other oxidants, disulfide bond equivalents of PDI can be used to form disulfide bonds in RNase in a stoichiometric reaction. In the absence of a glutathione redox buffer, the rate of reduced ribonuclease regeneration increases markedly with increasing PDI concentrations below the equivalence point; however, PDI in excess over stoichiometric concentrations inhibits RNase regneration.