Use of activators and inhibitors to define the properties of the active site of normal and Gaucher disease lysosomal β-glucosidase

S. Gatt, T. Dinur, K. Osiecki, R. J. Desnick, G. A. Grabowski

Research output: Contribution to journalArticlepeer-review

12 Scopus citations


Lysosomal β-glucosidase ('glucocerebrosidase') in peripheral blood lymphocyte and spleen extracts from normal individuals and Ashkenazi-Jewish Gaucher disease type-1 patients were investigated using several modifiers of glucosyl ceramide hydrolysis. The negatively charged lipids, phosphatidylserine and taurocholate, had differential effects on the hydrolytic rates of the normal and Gaucher disease enzymes from either source. With the normal enzyme, either negatively charged lipid (up to 1 mmol/l) increased the reaction rates, while decreasing hydrolytic rates were obtained at greater concentrations. In comparison, the peak activities of the Gaucher enzymes were observed at about 2-3 mmol/l or 5-8 mmol/l of phosphatidylserine or taurocholate, respectively. These negatively charged lipids altered only the velocity of the reactions; the apparent K(m) values were not affected. Taurocholate or phosphatidylserine also facilitated the interaction of the normal enzyme with conduritol B epoxide, a covalent inhibitor of the catalytic site. Compared to the normal enzyme, the Ashkenazi-Jewish Gaucher type-1 enzyme required about 5-fold greater concentrations of conduritol B epoxide for 50% inhibition. Neutral or cationic acyl-β-glucosides were found to be competitive or noncompetitive inhibitors of the enzymes, respectively. Alkyl β-glucosides were competitive (or linear-mixed type) inhibitors of the normal splenic or lymphocyte enzyme with competitive inhibition constants (K(i)) inversely related to the chain length. With octyl and dodecyl β-glucoside nearly normal competitive K(i) values were obtained with the splenic enzymes from Gaucher patients. The K(i) values were not influenced by increasing phosphatidylserine or taurocholate concentrations. In contrast, the cationic lipids, sphingosyl-1-O-β-D-glucoside (glucosyl sphingosine) and its N-hexyl derivative, were noncompetitive inhibitors whose apparent K(i) values for the normal enzyme were 30 and 0.25 μmol/l, respectively. The K(i) values for these sphingosyl glucosides were about increased 5 times for the Gaucher type-1 enzymes from Ashkenazi-Jewish Gaucher disease type-1 patients. The K(i) values of glucosyl sphingosine for the normal or mutant enzymes were directly related to increasing concentrations of phosphatidylserine or taurocholate. These data conform to the hypothesis that the active site of human lysosomal β-glucosidase is composed of at least three domains: a catalytic site which splits the β-glucosidic linkage; and 'aglycon binding site' which binds the ceramide (N-acyl-sphingosyl) residue of the substrate, and a third domain which interacts with the negatively charged lipids (e.g., phosphatidylserine) and the cationic sphingosyl glucosides, thereby altering enzyme catalysis by modulation of V(max) This latter site appears to be specifically altered by a mutation in the structural gene for lysosomal β-glucosidase in the Ashkenazi-Jewish form of type-1 Gaucher disease.

Original languageEnglish
Pages (from-to)109-119
Number of pages11
Issue number2
StatePublished - 1985


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