Vocadlo D J, Davies G J, Laine R, Withers S G
Protein Engineering Network of Centres of Excellence and the Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.
Nature. 2001 Aug 23;412(6849):835-8. doi: 10.1038/35090602.
Hen egg-white lysozyme (HEWL) was the first enzyme to have its three-dimensional structure determined by X-ray diffraction techniques. A catalytic mechanism, featuring a long-lived oxocarbenium-ion intermediate, was proposed on the basis of model-building studies. The 'Phillips' mechanism is widely held as the paradigm for the catalytic mechanism of beta-glycosidases that cleave glycosidic linkages with net retention of configuration of the anomeric centre. Studies with other retaining beta-glycosidases, however, provide strong evidence pointing to a common mechanism for these enzymes that involves a covalent glycosyl-enzyme intermediate, as previously postulated. Here we show, in three different cases using electrospray ionization mass spectrometry, a catalytically competent covalent glycosyl-enzyme intermediate during the catalytic cycle of HEWL. We also show the three-dimensional structure of this intermediate as determined by X-ray diffraction. We formulate a general catalytic mechanism for all retaining beta-glycosidases that includes substrate distortion, formation of a covalent intermediate, and the electrophilic migration of C1 along the reaction coordinate.
鸡蛋清溶菌酶（HEWL）是首个通过X射线衍射技术确定其三维结构的酶。基于模型构建研究，提出了一种以长寿命碳正离子中间体为特征的催化机制。“菲利普斯”机制被广泛认为是β-糖苷酶催化机制的范例，这类酶在切割糖苷键时能使异头中心的构型净保留。然而，对其他保留型β-糖苷酶的研究提供了有力证据，表明这些酶存在一种共同机制，涉及共价糖基-酶中间体，正如之前所假设的那样。在此，我们通过电喷雾电离质谱在三种不同情况下展示了HEWL催化循环过程中具有催化活性的共价糖基-酶中间体。我们还展示了通过X射线衍射确定的该中间体的三维结构。我们为所有保留型β-糖苷酶制定了一种通用催化机制，该机制包括底物变形、共价中间体的形成以及C1沿反应坐标的亲电迁移。
