Brandes H K, Larimer F W, Geck M K, Stringer C D, Schürmann P, Hartman F C
Protein Engineering Program, Oak Ridge-University of Tennessee Graduate School of Biomedical Science, Oak Ridge National Laboratory 37831.
J Biol Chem. 1993 Sep 5;268(25):18411-4.
Thioredoxin, by virtue of the proximal active-site sulfhydryls (Trp-Cys-Gly-Pro-Cys), catalyzes thiol-disulfide exchange with specific target enzymes. Considerable data (chemical modification, spectroscopic, and crystallographic) have implicated the cysteinyl residue nearest the N terminus of thioredoxin as the primary nucleophile; however, direct proof has been lacking. Proof is now provided by characterization of site-directed mutants of thioredoxin f with respect to activation of chloroplastic fructose-1,6-bisphosphatase (FBPase). The C49S mutant retains the capacity to activate FBPase, whereas the C46S mutant is totally lacking in this regard. Based on kinetics of FBPase activation, wild-type and C49S thioredoxins exhibit half-saturation values of 0.9 and 4 microM, respectively. Lack of activation by C46S is not because of failure to interact with FBPase, for it exhibits a Ki of 5 microM in competition with wild-type thioredoxin. Therefore, in the normal thioredoxin-catalyzed reduction pathway, Cys-46 is the nucleophile required to attack the disulfide of the substrate and Cys-49 serves to cleave the mixed disulfide intermediate, thus allowing for the release of oxidized thioredoxin and the reduced target enzyme.
硫氧还蛋白凭借其近端活性位点巯基（Trp-Cys-Gly-Pro-Cys）催化与特定靶酶的硫醇-二硫键交换。大量数据（化学修饰、光谱学和晶体学数据）表明，硫氧还蛋白N端附近的半胱氨酸残基是主要亲核试剂；然而，一直缺乏直接证据。现在通过对硫氧还蛋白f定点突变体激活叶绿体果糖-1,6-二磷酸酶（FBPase）的特性进行表征提供了证据。C49S突变体保留了激活FBPase的能力，而C46S突变体在这方面则完全缺乏。基于FBPase激活的动力学，野生型和C49S硫氧还蛋白的半饱和值分别为0.9和4 microM。C46S不能激活并非是因为无法与FBPase相互作用，因为它在与野生型硫氧还蛋白竞争时的Ki为5 microM。因此，在正常的硫氧还蛋白催化的还原途径中，Cys-46是攻击底物二硫键所需的亲核试剂，而Cys-49则用于裂解混合二硫键中间体，并释放氧化型硫氧还蛋白和还原型靶酶。
