Saturation-transfer-difference NMR to characterize substrate binding recognition and catalysis of two broadly specific glycoside hydrolases

摘要

Saturation-transfer-difference NMR spectroscopy(STD NMR)is used to delineate noncovalent enzyme-substrate interactions of beta-glycosidases from Pyrococcus furiosus and Aspergillus fumigatus under binding-only conditions at low temperatures,and during catalysis.Glucopyranosyl and galactopyranosyl moieties display a distinct pattern of multiple contacts with each active site,revealing enzyme-specific elements of recognition and portraying the global binding effect caused by single-site modification of the substrate,at carbon 4.The glucopyranose leaving group of cellobiose or lactose shows small relative STD effects except for the anomeric carbon,particularly in the alpha-form.Its replacement in beta-glucosides by an alcohol leaving group strongly affects sugar binding in the proximal enzyme subsite.A combination of STD effects of substrate and product,produced by the catalytic event or added exogenously,characterizes subsite binding during cellobiose hydrolysis.