Department of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712, United States.
ACS Chem Biol. 2012 Nov 16;7(11):1840-7. doi: 10.1021/cb300232n. Epub 2012 Aug 29.
The human asparaginase-like protein 1 (hASRGL1) catalyzes the hydrolysis of l-asparagine and isoaspartyl-dipeptides. As an N-terminal nucleophile (Ntn) hydrolase superfamily member, the active form of hASRGL1 is generated by an intramolecular cleavage step with Thr168 as the catalytic residue. However, in vitro, autoprocessing is incomplete (~50%), fettering the biophysical characterization of hASRGL1. We circumvented this obstacle by constructing a circularly permuted hASRGL1 that uncoupled the autoprocessing reaction, allowing us to kinetically and structurally characterize this enzyme and the precursor-like hASRGL1-Thr168Ala variant. Crystallographic and biochemical evidence suggest an activation mechanism where a torsional restraint on the Thr168 side chain helps drive the intramolecular processing reaction. Cleavage and formation of the active site releases the torsional restriction on Thr168, which is facilitated by a small conserved Gly-rich loop near the active site that allows the conformational changes necessary for activation.
人天冬酰胺酶样蛋白 1（hASRGL1）催化 l-天冬酰胺和异天冬酰二肽的水解。作为 N-末端亲核体（Ntn）水解酶超家族成员，hASRGL1 的活性形式通过 Thr168 作为催化残基的分子内切割步骤生成。然而，在体外，自体加工不完全（约 50%），限制了 hASRGL1 的生物物理特性的研究。我们通过构建一个环状排列的 hASRGL1 来绕过这个障碍，该 hASRGL1 使自体加工反应解偶联，从而使我们能够对该酶和前体样 hASRGL1-Thr168Ala 变体进行动力学和结构表征。晶体学和生化证据表明了一种激活机制，其中 Thr168 侧链上的扭转约束有助于驱动分子内加工反应。活性位点的切割和形成释放了 Thr168 上的扭转限制，这得益于靠近活性位点的小保守甘氨酸丰富环，该环允许激活所需的构象变化。
