β-Glucocerebrosidase is a member of the glycoside hydrolase family 30 and consists of three distinct domains. Domain I forms a three-stranded anti-parallel β-sheet. This domain contains two disulfide bridges that are necessary for correct folding, as well as a glycosylated residue that is required for catalytic activityin vivo. Domain II consists of two β-sheets that resemble an immunoglobulin fold. Domain III is homologous to a TIM barrel and is a highly conserved domain among glycoside hydrolases. Domain III harbors the active site, which binds the substrate glucocerebroside in close proximity to the catalytic residues E340 and E235. Domains I and III are tightly associated, while domains II and III are joined by a disordered linker.
Mechanism
s indicate that β-glucocerebrosidase binds the glucose moiety and adjacent O-glycosydic bond of glucocerebroside. The two aliphatic chains of glucocerebroside may remain associated with the lysosomal bilayer or interact with the activating protein Saposin C. Consistent with other glycoside hydrolases, the mechanism of glucocerebroside hydrolysis by β-glucocerebrosidase involves acid/base catalysis by two glutamic acid residues and precedes through a two-step mechanism. In the first step, E340 performs a nucleophilic attack at the carbon of the O-glycosidic linkage to displace the sphingosine moiety, which is simultaneously protonated by E235 as it is released from the active site. In the second step, glucose is hydrolyzed from the E340 residue to regenerate the active enzyme.
Properties
β-Glucocerebrosidase is maximally active at pH 5.5, the pH of the lysosomal compartment. Within the lysosome, GCase remains associated with the membrane, where it binds and degrades its substrate glucocerebroside. GCase requires the activating protein Saposin C as well as negatively charged lipids for maximal catalytic activity. The role of Saposin C is not known; however, it is shown to bind both the lysosomal membrane and the lipid moieties of GluCer, and therefore may recruit GluCer to the active site of the enzyme. β-Glucocerebrosidase is specifically and irreversibly inhibited by the glucose analog Conduritol B epoxide. Conduritol B epoxide binds to the GCase active site, where the enzyme cleaves its epoxide ring, forming a permanent covalent bond between the enzyme and the inhibitor. Initially, GCase was thought to be one of the fewlysosomal enzymes that does not follow the mannose-6-phosphate pathway for trafficking to the lysosome. A study in I-cell disease fibroblasts showed targeting of GCase to the lysosome independent of the M6P pathway. The lysosomal transporter and integral membrane proteinLIMP-2 was shown to bind GCase and facilitate transport to the lysosome, demonstrating a mechanism for M6P-independent lysosomal trafficking. This conclusion was called into question when a crystal structure of GCase in complex with LIMP-2 showed a Mannose 6-phosphate moiety on LIMP-2, suggesting the complex can also follow the traditional mannose-6-phosphate pathway.
was a version of glucocerebrosidase that was harvested from human placentaltissue and then modified with enzymes. It was approved by the FDA in 1991 and has been withdrawn from the market due to the approval of similar drugs made with recombinant DNA technology instead of being harvested from tissue. Drugs made recombinantly pose no risk of diseases being transmitted from the tissue used in harvesting, and are less expensive to manufacture. Recombinant glucocerebrosidases used as drugs include:
