Glucosidase II from rat liver microsomes. Kinetic model for binding and hydrolysis

Glucosidase II is an enzyme involved in glycoprotein biosynthesis, releasing both alpha-1,3-linked glucose residues from the protein-linked oligosaccharide Glc2Man9GlcNAc2-R in the processing of N-glycans. We studied the kinetic properties of the enzyme, purified to homogeneity and, for the first time, we have been able to demonstrate the occurrence of two active sites in this enzyme and to establish the mechanisms of binding and hydrolysis of the physiological substrate at its active site(s). The analyses of data fitting to single and double hyperbolic equations and the Eadie-Hofstee profile, together with the inhibition kinetics, demonstrate that the enzyme has two different active sites. The Km and Vmax. values for the high-affinity site (site 1) were 0.78 mM and 437 munits/mg respectively, whereas the values for the low-affinity site (site 2) were 481 mM and 13797 munits/mg respectively, for the p-nitrophenyl alpha-D-glucopyranoside substrate. The Vmax./Km ratios, which indicate the efficacy of an active site for a substrate, were 560 and 28.7 ml/min per g for active sites 1 and 2, respectively. The inhibition type, with respect to site 1, for glucose, maltose, D-glucone-delta-lactone, CaCl2 and MgCl2 was pure-competitive, partial-competitive, parabolic, non-competitive and non-competitive respectively. Ki values for glucose, maltose, CaCl2 and MgCl2 were 6.75, 2.05, 10.60 and 14.20 mM respectively. Thus glucose would bind to active site 1, maltose to site 2 (and near to site 1) and D-glucone-delta-lactone to either site 1 or 2. The following hydrolysis mechanism for the physiological substrate (Glc2Man9GlcNAc2-protein) of glucosidase II may be concluded from all the foregoing kinetic evidence: the external glucose would be the first released residue, at active site 2, thereafter producing Glc1Man9GlcNAc2-protein; the remaining glucose would be released at active site 1, delivering the Man9GlcNAc2-protein product, which would leave the enzyme.