Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA.
J Enzyme Inhib Med Chem. 2012 Dec;27(6):861-7. doi: 10.3109/14756366.2011.622272. Epub 2011 Oct 15.
The initial transcript of the GLS1 gene undergoes alternative splicing to produce two glutaminase variants (KGA and GAC) that contain unique C-terminal sequences. A truncated form of human glutaminase (hGA(124-551)) that lacks either C-terminal sequence was expressed in E.Coli and purified. This construct exhibits a hyperbolic glutamine saturation profile (K(m) of 1.6 mM). BPTES, bis-2[5-phenylacetamido-1,2,4-thiadiazol-2-yl]ethylsulfide, functions as a potent uncompetitive inhibitor of this construct (K(i) of 0.2 µM). The hGA(124-551) is inactive in the absence of phosphate, but exhibits a hyperbolic phosphate-dependent activation profile that is also inhibited by BPTES. Gel filtration studies indicate that hGA(124-551) forms a dimer in the absence or presence of 100 mM phosphate, whereas addition of BPTES causes the formation of an inactive tetramer. The combined data indicate that BPTES inhibits human glutaminase by a novel mechanism and that BPTES is a potential lead compound for development of an effective cancer chemotherapeutic agent.
GLS1 基因的初始转录本通过可变剪接产生两种谷氨酰胺酶变体（KGA 和 GAC），它们含有独特的 C 末端序列。在大肠杆菌中表达并纯化了一种缺乏任一 C 末端序列的人谷氨酰胺酶（hGA(124-551)）的截断形式。该构建体表现出双曲线谷氨酰胺饱和曲线（K(m)为 1.6mM）。BPTES，双-[5-（对乙酰氨基-1,2,4-噻二唑-2-基）乙硫醚，作为该构建体的有效非竞争性抑制剂起作用（K(i)为 0.2µM）。hGA(124-551)在没有磷酸盐的情况下没有活性，但表现出双曲线磷酸盐依赖性激活曲线，也被 BPTES 抑制。凝胶过滤研究表明，hGA(124-551)在没有或存在 100mM 磷酸盐的情况下形成二聚体，而添加 BPTES 导致无活性四聚体的形成。综合数据表明，BPTES 通过一种新的机制抑制人谷氨酰胺酶，并且 BPTES 是开发有效癌症化疗药物的潜在先导化合物。
