Zhang Wei, Modén Olof, Tars Kaspars, Mannervik Bengt
Department of Biochemistry and Organic Chemistry, Uppsala University, BMC, Box 576, SE-75123 Uppsala, Sweden.
Chem Biol. 2012 Mar 23;19(3):414-21. doi: 10.1016/j.chembiol.2012.01.021.
Glutathione transferase (GST) A2-2 is the most efficient human enzyme in the biotransformation of the prodrug azathioprine (Aza). The activation of Aza has therapeutic potential for possible use of GSTs in targeted enzyme-prodrug treatment of diseases. Based on the assumed catalytic mechanism and computational docking of Aza to the active site of the enzyme, active-site residues were selected for construction of focused mutant libraries, which were thereafter screened for Aza activity. Mutants with elevated Aza activity were identified, DNA sequenced, and the proteins purified. The two most active mutants showed up to 70-fold higher catalytic efficiency than the parental GST A2-2. The structure of the most active triple mutant (L107G/L108D/F222H) enzyme was determined by X-ray crystallography demonstrating significant changes in the topography of the active site facilitating productive binding of Aza as a substrate.
谷胱甘肽转移酶（GST）A2-2是前药硫唑嘌呤（Aza）生物转化过程中最有效的人类酶。Aza的激活对于在疾病的靶向酶-前药治疗中可能使用GST具有治疗潜力。基于假定的催化机制以及Aza与该酶活性位点的计算对接，选择活性位点残基构建聚焦突变体文库，随后对其进行Aza活性筛选。鉴定出具有升高的Aza活性的突变体，进行DNA测序并纯化蛋白质。两个活性最高的突变体的催化效率比亲本GST A2-2高出70倍。通过X射线晶体学确定了活性最高的三重突变体（L107G/L108D/F222H）酶的结构，结果表明活性位点的拓扑结构发生了显著变化，有利于Aza作为底物的有效结合。
