Klarskov K, Piens K, Ståhlberg J, Høj P B, Beeumen J V, Claeyssens M
Department of Biochemistry, Physiology and Microbiology, University of Gent, Belgium.
Carbohydr Res. 1997 Nov 10;304(2):143-54. doi: 10.1016/s0008-6215(97)00215-2.
(R,S)-3,4-Epoxybutyl beta-cellobioside, but not the corresponding propyl and pentyl derivatives, inactivates specifically and irreversibly cellobiohydrolase I from Trichoderma reesei by covalent modification of Glu212, the putative active-site nucleophile. The position and identity of the modified amino acid residue were determined using a combination of comparative liquid chromatography coupled on-line to electrospray ionization mass spectrometry, tandem mass spectrometry and microsequencing. It was found that the core protein corresponds to the N-terminal sequence pyrGlu1-Gly434 (Gly435) of intact cellobiohydrolase I. In the particular enzyme samples investigated, the asparagine residues in positions 45, 270 and 384 are each linked to a single 2-acetamido-2-deoxy-D-glucopyranose residue.
（R，S）-3,4-环氧丁基β-纤维二糖苷，而非相应的丙基和戊基衍生物，通过共价修饰假定的活性位点亲核试剂Glu212，特异性且不可逆地使里氏木霉的纤维二糖水解酶I失活。使用在线连接电喷雾电离质谱、串联质谱和微量测序的比较液相色谱法组合，确定了修饰氨基酸残基的位置和身份。发现核心蛋白对应于完整纤维二糖水解酶I的N端序列pyrGlu1-Gly434（Gly435）。在所研究的特定酶样品中，第45、270和384位的天冬酰胺残基各自与一个单一的2-乙酰氨基-2-脱氧-D-吡喃葡萄糖残基相连。
