Kinetic analysis of the interaction of alkyl glycosides with two human β-glucosidases

Abstract

This paper addresses the similarities and differences in the topology of the catalytic centres of human liver cytosolic β-glucosidase and placental lysosomal glucocerebrosidase, and utilizes well-documented reversible active-site-directed inhibitors. This comparative kinetic study was performed mainly to decipher the chemical and structural nature of the active site of the cytosolic β-glucosidase, whose physiological function is unknown. Specifically, analysis of the effects of a family of alkyl β-D-glucosides on the two glucosidases has led us to conclude that, relative to lysosomal glucocerebrosidase, the soluble β-glucosidase has a much more hydrophobic subsite in its catalytic centre. The alkyl β-D-glucosides consistently displayed 100-250-fold lower inhibition constants with the cytosolic broad-specificity β-glucosidase compared with the placental glucocerebrosidase; for example, with octyl β-D-glucoside the K(i) values were 10 μM and 1490 μM for the cytosolic and lysosomal β-glucosidases respectively. Furthermore the higher affinity of the cytosolic β-glucosidase than glucocerebrosidase for the amphipathic alkyl β-D-glucosides was validated by the greater increase in the free energy of binding with increasing alkyl chain length [ΔΔG(o)(K(i))/CH2: lysosomal enzyme, 2.01 kJ/mol (480 cal/mol); cytosolic enzyme, 3.05 kJ/mol (730 cal/mol)]. The implications of the presence of highly non-polar domains in the active site of the cytosolic β-glucosidase are discussed with regard to its potential physiological substrates.