Characterization of calf liver glucosidase I and its inhibition by basic sugar analogs.
Glucosidase I, the enzyme catalyzing the first step of N-linked oligosaccharide processing, has been purified from calf liver crude membranes [H. Hettkamp, G. Legler, and E. Bause, (1984) Eur. J. Biochem. 142, 85-90]. Binding experiments with concanavalin A-Sepharose suggest that glucosidase I is a glycoprotein with high-mannose carbohydrate chain(s). The enzyme has a subunit molecular mass of approximately 83 kDa and specifically hydrolyzes the terminal alpha-1,2-linked glucose residue from the natural Glc3-Man9-GlcNAc2 oligosaccharide. Studies with a variety of substrates modified in the aglycon moiety suggest that the Glc2 branch rather than the more distant domains of the substrate molecule are important for binding and hydrolysis. Glucosidase I does not require metal ions for activity and is strongly inhibited by 1-deoxynojirimycin (dNM) and its N-alkyl derivatives. Ki values range from 0.07 microM for N-methyl-dNM to 1.0 microM for dNM, measured at the pH-optimum of enzyme activity. The pH dependence of inhibition indicates that the cationic form of the inhibitors is the active species. Comparison of the Ki for N-decanoyl-dNM (approximately 70 microM) with that of N-decyl-dNM (approximately 0.4 microM) suggests that electrostatic interactions at the catalytic site of the enzyme are important for inhibitor binding. 1-Deoxymannojirimycin, previously assumed to be a specific mannosidase inhibitor, as well as its N-methyl and N-5-carboxypentyl derivatives, inhibit glucosidase I with Ki values around 190, 17, and 100 microM, respectively. This apparent lack of specificity shows that in vivo experiments on N-glycoprotein processing as well as the interpretation of results with these mannosidase inhibitors may give misleading results when these compounds are used in the millimolar range.
D017485:1-Deoxynojirimycin; D000818:Animals; D002417:Cattle; D005944:Glucosamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D005959:Glucosidases / Q000378:metabolism*; D065089:Glycoside Hydrolase Inhibitors; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D013379:Substrate Specificity; D000520:alpha-Glucosidases / Q000378:metabolism*
D065089:Glycoside Hydrolase Inhibitors; D009844:Oligosaccharides; D017485:1-Deoxynojirimycin; D005959:Glucosidases; C042059:glucosidase I; D000520:alpha-Glucosidases; D005944:Glucosamine