期刊
CARBOHYDRATE RESEARCH
卷 329, 期 3, 页码 539-547出版社
ELSEVIER SCI LTD
DOI: 10.1016/S0008-6215(00)00214-7
关键词
5-fluoro-alpha-D-galactopyranosyl fluoride; slow substrate; alpha-galactosidase; covalent glycosyl-enzyme intermediate; mass spectrometry
资金
- NHLBI NIH HHS [HL55482-01A1] Funding Source: Medline
5-Fluoro-alpha -D-galactopyranosyl fluoride was synthesized and its interaction with the active site of an alpha -galactosidase from green coffee bean (Coffea arabica), a retaining glycosidase, characterized kinetically and structurally. The compound behaves as an apparently tight binding (K-i = 600 nM) competitive inhibitor, achieving this high affinity through reaction as a slow substrate that accumulates a high steady-state concentration of the glycosyl-enzyme intermediate, as evidenced by ESiMS. Proteolysis of the trapped enzyme coupled with HPLC/MS analysis allowed the localization of a labeled peptide that was subsequently sequenced. Comparison of this sequence information to that of other members of the same glycosidase family revealed the active site nucleophile to be Asp145 within the sequence LKYDNCNNN. The importance of this residue to catalysis has been confirmed by mutagenesis studies. (C) 2000 Elsevier Science Ltd. All rights reserved.
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