Department of Pathology, Stanford University School of Medicine, Stanford, California 94305-5324, USA.
Protein Sci. 2011 Dec;20(12):1991-2003. doi: 10.1002/pro.741. Epub 2011 Nov 1.
The existence of low levels of intersubunit communication in homooligomeric enzymes is often difficult to discover, as the identical active sites cannot be probed individually to dissect their interdependent contributions. The homodimeric paralogs, E. coli aspartate- (AATase) and tyrosine aminotransferase (TATase), have not been demonstrated to show allostery. To address this question, we engineered a hybrid aminotransferase containing two distinct catalytic pockets: an AATase and a TATase site. The TATase/AATase hybrid was constructed by grafting an engineered TATase active site into one of the catalytic pockets of E. coli AATase. Each active site conserves its specific catalytic and inhibitor binding properties, and the hybrid catalyzes simultaneously each aminotransferase reaction at the respective site. Importantly, association of a selective inhibitor into one of the catalytic pockets decreases the activity of the second active site by up to 25%, thus proving unequivocally the existence of allosteric communication between active sites. The procedure may be applicable to other homologous sets of enzymes.
同寡聚酶中亚基间通讯水平低的存在通常很难被发现，因为无法单独探测相同的活性部位来剖析它们相互依赖的贡献。同源二聚体的同工酶，大肠杆菌天冬氨酸（AATase）和酪氨酸转氨酶（TATase），尚未被证明具有变构作用。为了解决这个问题，我们设计了一种含有两个不同催化口袋的杂种转氨酶：一个 AATase 和一个 TATase 位点。TATase/AATase 杂种是通过将工程化的 TATase 活性部位嫁接到大肠杆菌 AATase 的一个催化口袋中构建的。每个活性部位都保留其特定的催化和抑制剂结合特性，杂种同时在各自的部位催化每个转氨酶反应。重要的是，将选择性抑制剂结合到一个催化口袋中会使第二个活性部位的活性降低多达 25%，从而明确证明活性部位之间存在变构通讯。该程序可能适用于其他同源酶组。
