通过捕获5-氟糖基-酶中间体鉴定黑曲霉31家族α-葡萄糖苷酶的催化亲核试剂。
Identification of the catalytic nucleophile of the Family 31 alpha-glucosidase from Aspergillus niger via trapping of a 5-fluoroglycosyl-enzyme intermediate.
作者信息
Lee S S, He S, Withers S G
机构信息
Department of Chemistry, University of British Columbia, Vancouver, BC, Canada V6T 1Z1.
出版信息
Biochem J. 2001 Oct 15;359(Pt 2):381-6. doi: 10.1042/0264-6021:3590381.
Abstract
The mechanism-based reagent 5-fluoro-alpha-d-glucopyranosyl fluoride (5F alpha GlcF) was used to trap a glycosyl-enzyme intermediate and identify the catalytic nucleophile at the active site of Aspergillus niger alpha-glucosidase (Family 31). Incubation of the enzyme with 5F alpha GlcF, followed by peptic proteolysis and comparative liquid chromatography/MS mapping allowed the isolation of a labelled peptide. Fragmentation analysis of this peptide by tandem MS yielded the sequence WYDMSE, with the label located on the aspartic acid residue (D). Comparison with the known protein sequence identified the labelled amino acid as Asp-224 of the P2 subunit.
摘要
基于机制的试剂5-氟-α-D-吡喃葡萄糖基氟化物（5FαGlcF）用于捕获糖基酶中间体，并鉴定黑曲霉α-葡萄糖苷酶（31家族）活性位点处的催化亲核试剂。将该酶与5FαGlcF一起孵育，随后进行胃蛋白酶消化和比较液相色谱/质谱图谱分析，从而分离出一个标记肽段。通过串联质谱对该肽段进行碎片分析，得到序列WYDMSE，标记位于天冬氨酸残基（D）上。与已知蛋白质序列进行比较，确定标记的氨基酸为P2亚基的Asp-224。
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