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Functional studies of rat galactokinase

机译:大鼠半乳糖激酶的功能研究

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Galactokinase is an ATP-dependent enzyme that catalyzes the phosphorylation of galactose to form galactose-1-phosphate. The defect in human galactokinase can result in the disease of galactosemia. On the other hand, the control of galactose-1-phosphate production by inhibiting galactokinase is a potential therapy for another disease referred to as classic galactosemia. Many pharmaceutically important compounds derive from carbohydrate-containing natural products, and glycorandomization is one of the most efficient approaches for complex secondary metabolites. Therefore, it is important to further understand the interaction between galactokinase and its substrate or substrate analogs. In the present study, we cloned and purified both N- and C-terminal His-tagged rat galactokinase. We then constructed and purified a variety of variant enzymes, which were studied using kinetics with galactose and its analogs as substrates. We found that the binding of the ATP may induce conformational change to the enzyme so that the enzyme can bind galactose specifically. Asp186 was found to be a possible catalytic residue. The mutants were incubated with fluorescent trinitrophenyl-ATP for the characterization of their ATP binding sites. Various other substrate analogs, aminoglycosides and flavanoids were also tested and found to be competitive inhibitors of rat galactokinase.
机译:半乳糖激酶是一种ATP依赖型酶,催化半乳糖的磷酸化反应形成1-磷酸半乳糖。人半乳糖激酶的缺陷可能导致半乳糖血症。另一方面,通过抑制半乳糖激酶来控制1-磷酸半乳糖的产生是另一种被称为经典半乳糖血症的疾病的潜在疗法。许多药学上重要的化合物都来自含碳水化合物的天然产物,糖化随机化是复杂的次级代谢产物最有效的方法之一。因此,重要的是进一步了解半乳糖激酶及其底物或底物类似物之间的相互作用。在本研究中,我们克隆并纯化了N末端和C末端带有His标签的大鼠半乳糖激酶。然后,我们构建并纯化了多种变体酶,使用半乳糖及其类似物作为底物进行动力学研究。我们发现,ATP的结合可能会诱导该酶的构象变化,因此该酶可以特异性结合半乳糖。发现Asp186可能是催化残留物。将突变体与荧光三硝基苯基-ATP孵育,以表征其ATP结合位点。还测试了多种其他底物类似物,氨基糖苷和类黄酮,它们是大鼠半乳糖激酶的竞争性抑制剂。

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