Crystallographic snapshot of a productive glycosylasparaginase-substrate complex
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.
| Medienart: |
Artikel |
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| Erscheinungsjahr: |
2007 |
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| Erschienen: |
2007 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:366 |
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| Enthalten in: |
Journal of molecular biology - 366(2007), 1 vom: 09. Feb., Seite 82-92 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Wang, Yeming [VerfasserIn] |
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| Themen: |
Amidohydrolases |
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| Anmerkungen: |
Date Completed 20.03.2007 Date Revised 27.12.2018 published: Print-Electronic PDB: 2GL9 Citation Status MEDLINE |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM167077880 |
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| 041 | |a eng | ||
| 100 | 1 | |a Wang, Yeming |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Crystallographic snapshot of a productive glycosylasparaginase-substrate complex |
| 264 | 1 | |c 2007 | |
| 336 | |a Text |b txt |2 rdacontent | ||
| 337 | |a ohne Hilfsmittel zu benutzen |b n |2 rdamedia | ||
| 338 | |a Band |b nc |2 rdacarrier | ||
| 500 | |a Date Completed 20.03.2007 | ||
| 500 | |a Date Revised 27.12.2018 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a PDB: 2GL9 | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, N.I.H., Extramural | |
| 650 | 7 | |a Amidohydrolases |2 NLM | |
| 650 | 7 | |a EC 3.5.- |2 NLM | |
| 650 | 7 | |a N-terminal nucleophile hydrolase |2 NLM | |
| 650 | 7 | |a EC 3.5.1.- |2 NLM | |
| 650 | 7 | |a Aspartylglucosylaminase |2 NLM | |
| 650 | 7 | |a EC 3.5.1.26 |2 NLM | |
| 700 | 1 | |a Guo, Hwai-Chen |e verfasserin |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t Journal of molecular biology |d 1960 |g 366(2007), 1 vom: 09. Feb., Seite 82-92 |w (DE-627)NLM000006777 |x 1089-8638 |7 nnns |
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