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. Legier, 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 α-1,2-linked glucose residue from the natural Glc 3 Man 9-GlcNAc 2 oligosaccharide. Studies with a variety of substrates modified in the aglycon moiety suggest that the Glc 2 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. K i values range from 0.07 μM for N-methyl-dNM to 1.0 μM 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 K i for N-decanoyl-dNM (~70 μM) with that of N-decyl-dNM (~0.4 μM) 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 K i values around 190, 17, and 100 μM, 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.