Use of Activators and Inhibitors to Define the Properties of the Active Site of Normal and Gaucher Disease Lysosomal ß-Glucosidase

Abstract

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.

Human acid beta-glucosidase: isolation and amino acid sequence of a peptide containing the catalytic site.

“…Previous studies of human acid P-glucosidase have provided information concerning physical and kinetic properties (6,(8)(9)(10)(11)(12)(13) and processing (14) of this lysosomal hydrolase in normal and Gaucher disease tissues. However, until the recent report of a complete cDNA sequence (20), little structural information had been available for the normal acid 3-glucosidase.…”

“…This membrane-associated glycoprotein is a homomer whose mature glycosylated subunit has a Mr of 67,000-73,000 (3)(4)(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)(7)(8)(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) that interacts with negatively charged lipids to increase hydrolytic rates or cationic sphingosyl moieties of enzyme inhibitors (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) that interacts with negatively charged lipids to increase hydrolytic rates or cationic sphingosyl moieties of enzyme inhibitors (9). It has been proposed that these three domains function in the binding and orientation of substrates and release of products, respectively (8,9). Defects of acid ,B-glucosidase function and/or processing and stability lead to the accumulation of GC and the resultant subtypes and variants of Gaucher disease, an inherited lysosomal storage disease (11)(12)(13)(14).…”

Saposin C Is Required for Normal Resistance of Acid β-Glucosidase to Proteolytic Degradation

“…Clearly, small amounts of PS expression were required to re-establish GCase activity. Importantly for these studies, the detergent assays used for estimating GCase in vitro activity do not require negatively charged phospholipids or saposin C. Indeed, the detergents (taurocholate and Triton X-100) replace the function of these two more natural activators and provide an assessment of the maximal catalytic power of GCase in cells (25). Thus, the activities are not related to the in vivo levels but rather reflect the level of catalytically active GCase within cells.…”

“…Saposin D stimulates partially purified acid ceramidase for the degradation of ceramide (24). Importantly, selected nonphysiologic detergentbased assays can obviate the need for negatively charged phospholipids and/or saposins in these GCase assays and can provide estimates of its total catalytic power within cells (25).…”