Hederos Sofia, Baltzer Lars
IFM Chemistry, Division of Organic Chemistry, Linköping University SE-581 83, Linköping, Sweden.
Biopolymers. 2005 Dec 15;79(6):292-9. doi: 10.1002/bip.20351.
The acyl transfer reaction of S-glutathionyl benzoate (GSB) is catalyzed by a rationally designed mutant of human glutathione transferase A1-1, A216H. The catalyzed reaction proceeds via the formation of an acyl intermediate and has been studied in the presence of nitrogen, oxygen, and sulfur nucleophiles to determine the selectivity with regards to nucleophile structure. Methanol was previously shown to react with the acyl intermediate and form the corresponding ester, methylbenzoate, under a significant rate enhancement. In the present investigation, the dependence on nucleophile structure and reactivity has been investigated. Ethane thiol gave rise to a larger rate enhancement in the enzyme-catalyzed reaction than ethanol, whereas ethylamine did not increase the reaction rate. The reactivities toward the acyl intermediate of primary and secondary alcohols with similar pKa values depended on the structure of the aliphatic chain, and 1-propanol was the most efficient alcohol. The reactivity of the oxygen nucleophiles was also found to depend strongly on pKa as 2,2,2-trifluoroethanol, with a pKa of 12.4, was the most efficient nucleophile of all that were tested. Saturation kinetics was observed in the case of 1-propanol, indicating a second binding site in the active site of A216H. The nucleophile selectivity of A216H provides the knowledge base needed for the further reengineering of A216H towards alternative substrate specificities.
人谷胱甘肽转移酶A1-1的合理设计突变体A216H催化S-谷胱甘肽基苯甲酸酯（GSB）的酰基转移反应。催化反应通过酰基中间体的形成进行，并且已经在氮、氧和硫亲核试剂存在下进行了研究，以确定对亲核试剂结构的选择性。先前已证明甲醇与酰基中间体反应并在显著的速率增强下形成相应的酯苯甲酸甲酯。在本研究中，研究了对亲核试剂结构和反应性的依赖性。乙硫醇在酶催化反应中比乙醇产生更大的速率增强，而乙胺没有提高反应速率。具有相似pKa值的伯醇和仲醇对酰基中间体的反应性取决于脂肪链的结构，1-丙醇是最有效的醇。还发现氧亲核试剂的反应性强烈依赖于pKa，因为pKa为12.4的2,2,2-三氟乙醇是所有测试亲核试剂中最有效的。在1-丙醇的情况下观察到饱和动力学，表明A216H活性位点存在第二个结合位点。A216H的亲核试剂选择性为进一步改造A216H以获得替代底物特异性提供了所需的知识库。
