State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
Biol Chem. 2012 Sep;393(9):1013-24. doi: 10.1515/hsz-2012-0183.
Arginine deiminase (ADI) is a potential antitumor agent for the arginine deprivation treatment of L-arginine auxotrophic tumors. The optimum pH of ADI varies significantly, yet little is known about the origin of this variety. Here, Pseudomonas aeruginosa ADI (PaADI), an enzyme that functions only at acidic pH, was utilized as the model system. The results of UV-pH titration imply that the nucleophilic Cys406 thiol group is protonated in the resting state. The H405R single mutation resulted in an altered pH optimum (from pH 5.5 to 6.5), an increased k(cat) (from 9.8 s(-1) to 101.7 s(-1) at pH 6.5), and a shifted pH rate dependence (ascending limb pK(a) from 3.6 to 4.4). Other mutants were constructed to investigate the effects of hydrogen bonding, charge distribution, and hydrophobicity on the properties of the enzyme. The pH optima of His405 mutants were all shifted to a relatively neutral pH except for the H405E mutant. The results of kinetic characterizations and molecular dynamic simulations revealed that the active site hydrogen bonding network involving Asp280 and His405 plays an important role in controlling the dependence of PaADI activity on pH. Moreover, the H405R variant showed increased cytotoxicity towards arginine auxotrophic cancer cell lines.
精氨酸脱亚氨酶（ADI）是一种潜在的抗肿瘤药物，可用于精氨酸剥夺治疗 L-精氨酸营养缺陷型肿瘤。ADI 的最适 pH 值变化很大，但对此种变异性的起源知之甚少。本研究以仅在酸性 pH 下发挥作用的铜绿假单胞菌 ADI（PaADI）为模型系统。紫外- pH 滴定结果表明，亲核 Cys406 巯基在静息状态下质子化。H405R 单点突变导致最适 pH 值发生改变（从 pH5.5 变为 pH6.5），kcat 增加（在 pH6.5 时，从 9.8s-1 增加至 101.7s-1），pH 速率依赖性发生改变（上升支 pK(a)从 3.6 变为 4.4）。进一步构建其他突变体以研究氢键、电荷分布和疏水性对酶性质的影响。除 H405E 突变体外，His405 突变体的最适 pH 值均向相对中性 pH 值偏移。动力学特征分析和分子动力学模拟的结果表明，涉及 Asp280 和 His405 的活性位点氢键网络在控制 PaADI 活性对 pH 的依赖性方面发挥着重要作用。此外，H405R 变体对精氨酸营养缺陷型癌细胞系表现出更高的细胞毒性。
