Kinetic characterization and identification of the acylation and glycosylation sites of recombinant human gamma-glutamyltranspeptidase.
Kinetic characterization and identification of the acylation and glycosylation sites of recombinant human gamma-glutamyltranspeptidase.
复制标题
重组人γ-谷氨酰转肽酶的酰化和糖基化位点的动力学表征和鉴定。
作者：
R. Castonguay;Dany Halim;Mylène Morin;A. Furtos;C. Lherbet;É. Bonneil;P. Thibault;J. Keillor
γ -谷氨酰转肽酶（GGT）是一种异二聚体酶，对谷胱甘肽稳态控制很重要。它还与许多生理疾病有关，包括帕金森病、细胞凋亡抑制和糖尿病。在其乒乓机制的第一步，它结合体内底物谷胱甘肽，并在形成酰基酶中间体时释放半胱氨酸。然后这种中间体可以与水反应释放谷氨酸作为水解产物，或者与氨基酸或二肽反应形成转肽化产物。对这些反应机制的进一步详细研究，至少对于某些ggt来说，由于从组织中经过多步骤纯化后可获得的蛋白质数量很少，因此受到阻碍。本研究将人GGT基因克隆到pPICZalphaA载体中，转化到毕赤酵母中表达为68 kDa的his标记蛋白。优化后的酶在1 L培养基中表达和分泌，固定化金属亲和层析纯化得到1.6 mg的纯化酶，比活性为237 U/mg。质谱法测定了谷胱甘肽和甘氨酸之间转肽化反应的动力学参数，kcat为13.4 × 10(3) min-1，表观KM值谷胱甘肽为1.11 mM，甘氨酸为8.1 mM。还研究了ggt介导的谷胱甘肽水解，提供了53 min-1的kcat和谷胱甘肽的KM值为7.3微米。该酶与基于机制的抑制剂、酶消化和质谱分析的孵育首次明确鉴定了Thr381是人类γ -谷氨酰转肽酶的活性位点亲核试剂，并确认了7个n链糖基化位点中的4个。这些结构和动力学数据讨论了相对于同源模型产生，以方便可视化。
Gamma-glutamyltranspeptidase (GGT) is a heterodimeric enzyme important for glutathione homeostasis control. It has also been implicated in many physiological disorders, including Parkinson's disease, apoptosis inhibition, and diabetes. In the first step of its ping-pong mechanism it binds glutathione, its in vivo substrate, and releases cysteinylglycine upon formation of an acyl-enzyme intermediate. This intermediate can then react with water to release glutamate as a hydrolysis product or with an amino acid or dipeptide to form a transpeptidation product. Further detailed study of the mechanism underlying these reactions is hindered at least for some GGTs by the low quantities of protein available after a multistep purification from tissue. In the present work the gene for human GGT was cloned into the pPICZalphaA vector and transformed into Pichia pastoris to express as a 68 kDa His-tagged protein. The optimized expression and secretion of this enzyme in 1 L of culture and subsequent purification by immobilized metal affinity chromatography yielded 1.6 mg of purified enzyme having a specific activity of 237 U/mg. Kinetic parameters for the transpeptidation reaction between glutathione and glycylglycine were determined by mass spectrometry, giving a kcat of 13.4 x 10(3) min-1 and apparent KM values of 1.11 mM for glutathione and 8.1 mM for glycylglycine. The GGT-mediated hydrolysis of glutathione was also studied, providing a kcat of 53 min-1 and a KM value of 7.3 microM for glutathione. Incubation of the enzyme with a mechanism-based inhibitor, enzymatic digest, and mass spectrometric analysis provided the first unambiguous identification of Thr381 as the active site nucleophile of human gamma-glutamyltranspeptidase, and confirmed four of the seven N-linked glycosylation sites. These structural and kinetic data are discussed with respect to a homology model generated to facilitate visualization.