利用6-磷酸-β-葡萄糖苷酶在低温条件下获得酶-底物复合物实际结构的方法

A method to determine the structure of the complex of enzyme and its substrate using 6-phosphate-β-glucosidase at low temperature

利用"酶晶体低温抑活底物固定"方法,在-20℃条件下,将6-磷酸-β-葡萄糖苷酶(BglA)晶体与底物对硝基苯-β-D-吡喃葡萄糖苷-6-磷酸(pNPβG6P)进行浸泡试验,通过X射线进行衍射数据收集并处理,获得了完整的底物电子密度图。研究结果表明,在不突变酶中关键基团使酶失活,以及不选用底物类似物替代原有底物的情况下,通过上述方法,可以获得酶与底物复合物的实际结构。该研究结果可为今后低温酶学及复合物中间态的进一步研究提供帮助。

To capture a state of the enzyme in complex with an intact substrate, we developed and adopted a novel freezing method in crystal preparation procedure. Neither the elimination of the catalytically indispensable ligands, nor mutation or modification of the active site is required. At -20 ℃, we soaked the crystal of 6-phosphate-β-glucosidase （BglA） in the liquor containing p-nitrophenyl-β-D-glucopyranoside-6-phosphate （pNPI3G6P）. The ,：liffraction data at 2 A resolution was collected and an intact and unambiguous electron density map of pNPI3G6P was obtained. These results provide an effective method for the research of cryoenzymology and the intermediate state of enzyme-substrate complex in the future.