β-Glucocerebrosidase (also called acid β-glucosidase, D-glucosyl-N-acylsphingosine glucohydrolase, or GCase) is an
enzyme with
glucosylceramidase activity (
EC3.2.1.45) that cleaves by
hydrolysis the
β-glycosidic linkage of the chemical
glucocerebroside, an intermediate in
glycolipid metabolism that is abundant in cell membranes (particularly skin cells).[5] It is localized in the
lysosome, where it remains associated with the lysosomal membrane.[6] β-Glucocerebrosidase is 497 amino acids in length and has a molecular mass of 59,700
Da.[citation needed]
Three-dimensional PyMol rendering of glucocerebrosidase with three domains highlighted.
Three-dimensional PyMol rendering of glucocerebrosidase with catalytic residues highlighted.
Domain I (residues 1–27 and 383–414) 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 (Asn19) that is required for catalytic activity in vivo. Domain II (residues 30–75 and 431–497) consists of two β-sheets that resemble an
immunoglobulin fold. Domain III (residues 76–381 and 416–430) is homologous to a
TIM barrel and is a highly conserved domain among
glycoside hydrolases.[8] 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.[7]
Consistent with other glycoside hydrolases, the mechanism of glucocerebroside
hydrolysis by β-glucocerebrosidase involves
acid/base catalysis by two
glutamic acid residues (E340 and E235) 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.[7][9]
Properties
β-Glucocerebrosidase is maximally active at pH 5.5, the pH of the lysosomal compartment.[10] Within the lysosome it remains associated with the membrane, where it binds and degrades its substrate
glucocerebroside (GluCer). It requires the activating protein
Saposin C as well as negatively charged lipids for maximal catalytic activity.[11][12] 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.[13][14]
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.[15]
Initially, GCase was thought to be one of the few lysosomal enzymes that does not follow the
mannose-6-phosphate pathway for trafficking to the
lysosome.[16] A study in
I-cell diseasefibroblasts (in which the
phosphotransferase that puts
Mannose 6-phosphate on proteins to target them to the lysosome is defective) showed targeting of GCase to the lysosome independent of the M6P pathway.[17] The lysosomal transporter and integral membrane protein
LIMP-2 (Lysosomal Integral Membrane Protein 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.[18]
Alglucerase (Ceredase) was a version of glucocerebrosidase that was harvested from human
placentaltissue and then modified with enzymes.[24] It was approved by the FDA in 1991[25] but has been withdrawn from the market[26][27] 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.[24]
Recombinant glucocerebrosidases used as drugs include:[28]
^Sinclair G, Pfeifer TA, Grigliatti TA, Choy FY (April 2006). "Secretion of human glucocerebrosidase from stable transformed insect cells using native signal sequences". Biochemistry and Cell Biology. 84 (2): 148–56.
doi:
10.1139/o05-165.
PMID16609695.
^Aerts JM, Sa Miranda MC, Brouwer-Kelder EM, Van Weely S, Barranger JA, Tager JM (October 1990). "Conditions affecting the activity of glucocerebrosidase purified from spleens of control subjects and patients with type 1 Gaucher disease". Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology. 1041 (1): 55–63.
doi:
10.1016/0167-4838(90)90122-V.
PMID2223847.
^Ogawa S, Uetsuki S, Tezuka Y, Morikawa T, Takahashi A, Sato K (June 1999). "Synthesis and evaluation of glucocerebrosidase inhibitory activity of anhydro deoxyinositols from (+)-epi- and (-)-vibo-quercitols". Bioorganic & Medicinal Chemistry Letters. 9 (11): 1493–8.
doi:
10.1016/S0960-894X(99)00223-1.
PMID10386923.
Fabrega S, Durand P, Mornon JP, Lehn P (2002). "[The active site of human glucocerebrosidase: structural predictions and experimental validations]". Journal de la Société de Biologie. 196 (2): 151–60.
doi:
10.1051/jbio/2002196020151.
PMID12360744.
S2CID81542873.