Human acid β-glucosidase: use of sphingosyl and N-alkyl-glucosylamine inhibitors to investigate the properties of the active site

Abstract

Human acid β-glucosidase ( d- glucosyl-N- acylsphingosine glucohydrolase, EC 3.2.1.45 ) cleaves the β-glucosidic bonds of glucosylceramide and synthetic β-glucosides. The specificity of binding to the active site of this enzyme was evaluated using series of inhibitors including synthetic sphingosines, N-alkyl(C n)-deoxynojirimycins (1,5-dideoxy-5-iminoglucose) and N-C n-glucosylamines. The sphingosines were rapidly reversible inhibitors with maximal potency ( IC 50 ≈ 78–150 μM) at chain lengths of 14–18 carbons. The presence of unsaturation between C4 and C5 was required for inhibition of enzyme activity. Neither the nature of this bond (double or triple bond) nor the presence of erythro or threo configurations at C2 influenced inhibitory potency. The N-C 10− to N-C 14-deoxynojirimycins were rapidly reversible inhibitors with K i ≈ 8.5 nM. In comparison, the 1-amino glucose derivatives, i.e., N-C n-glucosylamines (n = 12–18), were more potent ( IC 50 ≈ 0.3–3 nM) and their maximal inhibitory potencies were dependent on time as well as enzyme and substrate concentrations: i.e., the N-C 12− to N-C 18-glucosylamines were competitive, slow-tight binding inhibitors. Analyses of progress curves at various N-C n-glucosylamine (n = 14–18) concentrations indicated the formation of rapidly dissociating initial EI collison complex which then undergoes a conformational change to a slowly reversible EI ∗ complex. These results were consistent with the long chain N-C n-glucosylamines being reaction intermediate analogues and with this enzyme's hydrolytic mechanism requiring a conformational change during the transition state.