Shaikh Fathima Aidha, Müllegger Johannes, He Shouming, Withers Stephen G
Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada.
FEBS Lett. 2007 May 29;581(13):2441-6. doi: 10.1016/j.febslet.2007.04.053. Epub 2007 Apr 30.
The mechanism-based inhibitor 2,4-dinitrophenyl 2-deoxy-2-fluoro-beta-d-galactopyranoside (DNP2FGal) was used to inactivate the Family 42 beta-galactosidase (YesZ) from Bacillus subtilis via the trapping of a glycosyl-enzyme intermediate, thereby tagging the catalytic nucleophile in the active site. Proteolytic digestion of the inactivated enzyme and of a control sample of unlabeled enzyme, followed by comparative high-performance liquid chromatography and mass spectrometric analysis identified a labelled peptide of the sequence ETSPSYAASL. These data, combined with sequence alignments of this region with representative members of Family 42, unequivocally identify the catalytic nucleophile in this enzyme as Glu-295, thereby providing the first direct experimental proof of the identity of this residue within Family 42.
基于机制的抑制剂2,4-二硝基苯基2-脱氧-2-氟-β-D-吡喃半乳糖苷（DNP2FGal）用于通过捕获糖基酶中间体使枯草芽孢杆菌的42家族β-半乳糖苷酶（YesZ）失活，从而标记活性位点中的催化亲核试剂。对失活酶和未标记酶的对照样品进行蛋白水解消化，然后进行比较高效液相色谱和质谱分析，鉴定出序列为ETSPSYAASL的标记肽段。这些数据，结合该区域与42家族代表性成员的序列比对，明确确定该酶中的催化亲核试剂为Glu-295，从而提供了42家族中该残基身份的首个直接实验证据。
