Shah Luqman, Nadeem Muhammad Shahid, Khan Jalaluddin Azam, Zeyadi Mustafa A, Zamzami Mazin A, Mohammed Kaleemuddin
Department of Biochemistry, Faculty of Science, King Abdulaziz University Building A 90 Jeddah 21589 Saudi Arabia
RSC Adv. 2019 Feb 1;9(8):4258-4267. doi: 10.1039/c8ra04740e. eCollection 2019 Jan 30.
Glutaminase (GLS) is an enzyme essential for amino acid metabolism; in particular, it acts as a catalyst in glutaminolysis, a reaction exploited by the malignant cells to meet the nutrient requirements for their accelerated growth and proliferation. regulating the initial reaction of the glutaminolysis pathway, glutaminase offers an intriguing target for the development of anticancer drugs. In the present study, we produced a recombinant glutaminase from DSM-465 in . The enzyme was purified to electrophoretic homogeneity, with 40% recovery and 22.36 fold purity. It exhibited a molecular weight of 33 kDa, with an optimum pH and temperature of 9 and 70 °C, respectively. The value of the purified enzyme was 104 μM for l-glutamine. A 3D model was built for the enzyme using Swiss-Model and subjected to molecular docking with the substrate and potential inhibitors. Moreover, the subject enzyme was compared with the human kidney type GLS-K by ConSurf and TM-align servers for evolutionary conserved residues and structural domains. Despite having less than 40% amino acid identity, the superimposed monomers of both enzymes exhibited ∼94% structural identity. With a positional difference, the active site residues Ser65, Asn117, Glu162, Asn169, Tyr193, Tyr245, and Val263 found in the bacterial enzyme were also conserved in the human GLS-K. Molecular docking results have shown that CB-839 is the best inhibitor for GLS-GT and UPGL00004 is the best inhibitor for GLS-K, as designated by the binding free energy changes, Δ -388.7 kJ mol and Δ -375 kJ mol, respectively. Moreover, six potential inhibitory molecules were ranked according to their binding free energy change values for both enzymes. The information can be used for the anticancer studies.
谷氨酰胺酶（GLS）是氨基酸代谢所必需的一种酶；特别是，它在谷氨酰胺分解代谢中起催化剂作用，恶性细胞利用这一反应来满足其加速生长和增殖的营养需求。谷氨酰胺酶调节谷氨酰胺分解代谢途径的初始反应，为抗癌药物的开发提供了一个引人关注的靶点。在本研究中，我们从DSM - 465中制备了一种重组谷氨酰胺酶。该酶被纯化至电泳纯，回收率为40%，纯度提高了22.36倍。它的分子量为33 kDa，最适pH值和温度分别为9和70℃。纯化后的酶对L - 谷氨酰胺的Km值为104μM。使用Swiss - Model为该酶构建了三维模型，并与底物和潜在抑制剂进行分子对接。此外，通过ConSurf和TM - align服务器将该目标酶与人类肾脏型GLS - K进行比较，以确定进化保守残基和结构域。尽管两种酶的氨基酸同一性低于40%，但它们叠加的单体显示出约94%的结构同一性。在位置上存在差异的情况下，细菌酶中发现的活性位点残基Ser65、Asn117、Glu162、Asn169、Tyr193、Tyr245和Val263在人类GLS - K中也保守存在。分子对接结果表明，根据结合自由能变化，CB - 839是GLS - GT的最佳抑制剂，UPGL00004是GLS - K的最佳抑制剂，其结合自由能变化分别为Δ - 388.7 kJ/mol和Δ - 375 kJ/mol。此外，根据六种潜在抑制分子对两种酶的结合自由能变化值进行了排序。这些信息可用于抗癌研究。
