Department of Biological Sciences, National University of Singapore, Singapore.
Department of Biochemistry, Yong Loo Ling School of Medicine, National University of Singapore, Singapore.
FEBS J. 2023 May;290(9):2437-2448. doi: 10.1111/febs.16658. Epub 2022 Nov 9.
Glutaminase catalyses the metabolic process called glutaminolysis. Cancer cells harness glutaminolysis to increase energy reserves under stressful conditions for rapid proliferation. Glutaminases are upregulated in many tumours. In humans, the kidney-type glutaminase (KGA) isoform is highly expressed in the kidney, brain, intestine, foetal liver, lymphocytes and in many tumours. Glutaminase inhibition is shown to be effective in controlling cancers. Previously, we and others reported the inhibition mechanism of KGA using various inhibitors that target the active and allosteric sites of the enzyme. Here, we report the identification of a novel allosteric site in KGA using the compound DDP through its complex crystal structure combined with mutational and hydrogen-deuterium exchange mass spectrometry studies. This allosteric site is located at the dimer interface, situated ~ 31 Å away from the previously identified allosteric site and ~ 32 Å away from the active site. Remarkably, the mechanism of inhibition is conserved, irrespective of which allosteric pocket is targeted, causing the same conformational changes in the key loop near the active site (Glu312-Pro329) and subsequent enzyme inactivation. Contrary to the previously identified allosteric site, the identified new allosteric site is primarily hydrophilic. This site could be effectively targeted for the synthesis of specific and potent water-soluble inhibitors of glutaminase, which will lead to the development of anticancer drugs.
谷氨酰胺酶催化称为谷氨酰胺分解的代谢过程。癌细胞利用谷氨酰胺分解在应激条件下增加能量储备，以快速增殖。许多肿瘤中谷氨酰胺酶上调。在人类中，肾型谷氨酰胺酶（KGA）同工型在肾脏、大脑、肠道、胎肝、淋巴细胞和许多肿瘤中高度表达。谷氨酰胺酶抑制被证明在控制癌症方面是有效的。以前，我们和其他人使用靶向酶的活性和变构位点的各种抑制剂报告了 KGA 的抑制机制。在这里，我们通过复合物晶体结构结合突变和氘氚交换质谱研究，报告了使用化合物 DDP 在 KGA 中鉴定出一个新的变构位点。该变构位点位于二聚体界面，距离先前鉴定的变构位点约 31Å，距离活性位点约 32Å。值得注意的是，抑制机制是保守的，无论靶向哪个变构口袋，都会导致活性位点附近关键环的相同构象变化（Glu312-Pro329）和随后的酶失活。与先前鉴定的变构位点不同，鉴定出的新变构位点主要是亲水的。该位点可有效靶向合成特定且有效的水溶性谷氨酰胺酶抑制剂，从而开发出抗癌药物。
