Caner Sami, Zhang Xiaohua, Jiang Jianbing, Chen Hong-Ming, Nguyen Nham T, Overkleeft Hermen, Brayer Gary D, Withers Stephen G
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada.
Department of Chemistry, Faculty of Science, University of British Columbia, Vancouver, BC, Canada.
FEBS Lett. 2016 Apr;590(8):1143-51. doi: 10.1002/1873-3468.12143. Epub 2016 Apr 3.
As part of a search for selective, mechanism-based covalent inhibitors of human pancreatic α-amylase we describe the chemoenzymatic synthesis of the disaccharide analog α-glucosyl epi-cyclophellitol, demonstrate its stoichiometric reaction with human pancreatic α-amylase and evaluate the time dependence of its inhibition. X-ray crystallographic analysis of the covalent derivative so formed confirms its reaction at the active site with formation of a covalent bond to the catalytic nucleophile D197. The structure illuminates the interactions with the active site and confirms OH4' on the nonreducing end sugar as a good site for attachment of fluorescent tags in generating probes for localization and quantitation of amylase in vivo.
作为寻找人胰腺α-淀粉酶基于机制的选择性共价抑制剂的一部分，我们描述了二糖类似物α-葡萄糖基表环戊烯醇的化学酶法合成，证明了其与人胰腺α-淀粉酶的化学计量反应，并评估了其抑制作用的时间依赖性。对如此形成的共价衍生物的X射线晶体学分析证实了其在活性位点的反应，与催化亲核试剂D197形成了共价键。该结构阐明了与活性位点的相互作用，并确认了非还原端糖上的OH4'是连接荧光标签以生成用于体内淀粉酶定位和定量探针的良好位点。
