Human acid /3-glucosidase (D-glucosyl-Nacylsphingosine glucohydrolase, EC 3.2.1.45) cleaves the glucosidic bonds of glucosylceramide and synthetic aglucosides. The deficient activity of this hydrolase is the enzymatic defect in the subtypes and variants of Gaucher disease, the most prevalent lysosomal storage disease. To isolate and characterize the catalytic site of the normal enzyme, brominated 3H-labeled conduritol B epoxide (3H-Br-CBE), which inhibits the enzyme by binding covalently to this site, was used as an affinity label. Under optimal conditions 1 mol of 3H-Br-CBE bound to 1 mol of pure enzyme protein, indicating the presence of a single catalytic site per enzyme subunit. After V8 protease digestion of the 3H-Br-CBE-labeled homogeneous enzyme, three radiolabeled peptides, designated peptide A, B, or C, were resolved by reverse-phase HPLC. The partial amino acid sequence (37 residues) of peptide A (Mr, 5000) was determined. The sequence of this peptide, which contained the catalytic site, had exact homology to the sequence near the carboxyl terminus of the protein, as predicted from the nucleotide sequence of the full-length cDNA encoding acid /3-glucosidase.Human acid ,-glucosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45), a lysosomal enzyme, cleaves the /3-glucosyl linkage in glucosylceramide (GC) as well as synthetic P3-glucosides (1,2). This membrane-associated glycoprotein is a homomer whose mature glycosylated subunit has a Mr of 67,000-73,000 (3-5). The enzyme is hydrophobic and requires detergents, negatively charged lipids, and/or a "co-glucosidase" for optimal hydrolysis of GC or synthetic substrates (6-9). Detailed studies of the effects and interactions of a variety of enzyme modifiers have indicated that the active site of the enzyme contains at least three domains with differing specificities: (i) the catalytic site, a hydrophilic pocket that recognizes P3-glucosyl moieties and conduritol B epoxide (CBE); (ii) an aglycon binding site that is hydrophobic and has affinity for the alkyl chains ofGC; and (iii) a hydrophobic third domain (9) or "allosteric" site (10)

Lysosomal ß-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/1) 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/1 or 5-8 mmol/1 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-ß-glucosides were found to be competitive or noncompetitive inhibitors of the enzymes, respectively. Alkyl ß-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 ß-glucoside nearly normal competitive K(i) values were obtained with the splenic enzymes from Gaucher patients. These K(i) values were not influenced by increasing phosphatidylserine or taurocholate concentrations. In contrast, the cationic lipids, sphingosyl-l-Oß- 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 pmol/1, 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 ß-glucosidase is composed of at least three domains: (l)a catalytic site which splits the ß-glucosidic linkage; (2) an ‘aglycon binding site’ which binds the ceramide (N-acyl-sphingosyl) residue of the substrate, and (3) 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 ß-glucosidase in the Ashkenazi-Jewish form of type-1 Gaucher disease.