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

Abstract:

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 (pNPβG6P). The diffraction data at 2 ? resolution was collected and an intact and unambiguous electron density map of pNPβG6P was obtained. These results provide an effective method for the research of cryoenzymology and the intermediate state of enzyme-substrate complex in the future.