title: Crystallographic snapshot of a productive glycosylasparaginase-substrate complex.
abstract: Glycosylasparaginase (GA) plays an important role in asparagine-linked glycoprotein degradation. A deficiency in the activity of human GA leads to a lysosomal storage disease named aspartylglycosaminuria. GA belongs to a superfamily of N-terminal nucleophile hydrolases that autoproteolytically generate their mature enzymes from inactive single chain protein precursors. The side-chain of the newly exposed N-terminal residue then acts as a nucleophile during substrate hydrolysis. By taking advantage of mutant enzyme of Flavobacterium meningosepticum GA with reduced enzymatic activity, we have obtained a crystallographic snapshot of a productive complex with its substrate (NAcGlc-Asn), at 2.0 A resolution. This complex structure provided us an excellent model for the Michaelis complex to examine the specific contacts critical for substrate binding and catalysis. Substrate binding induces a conformational change near the active site of GA. To initiate catalysis, the side-chain of the N-terminal Thr152 is polarized by the free alpha-amino group on the same residue, mediated by the side-chain hydroxyl group of Thr170. Cleavage of the amide bond is then accomplished by a nucleophilic attack at the carbonyl carbon of the amide linkage in the substrate, leading to the formation of an acyl-enzyme intermediate through a negatively charged tetrahedral transition state.
authors.affiliation: Department of Physiology and Biophysics, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118-2526, USA.
authors.forename: Yeming
authors.initials: Y
authors.lastname: Wang
authors.forename: Hwai-Chen
authors.initials: HC
authors.lastname: Guo
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry; D001227:Aspartylglucosylaminase / Q000737:chemistry*; D001665:Binding Sites; D002384:Catalysis; D045247:Chryseobacterium / Q000737:chemistry; D018360:Crystallography, X-Ray / Q000379:methods*; D015394:Molecular Structure; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D000581:Amidohydrolases; C438834:N-terminal nucleophile hydrolase; D001227:Aspartylglucosylaminase