National Bioenergy Center and §Biosciences Center, National Renewable Energy Laboratory , Golden, Colorado 80401, United States.
J Am Chem Soc. 2014 Jan 8;136(1):321-9. doi: 10.1021/ja410291u. Epub 2013 Dec 16.
Glycoside hydrolases (GHs) cleave glycosidic linkages in carbohydrates, typically via inverting or retaining mechanisms, the latter of which proceeds via a two-step mechanism that includes formation of a glycosyl-enzyme intermediate. We present two new structures of the catalytic domain of Hypocrea jecorina GH Family 7 cellobiohydrolase Cel7A, namely a Michaelis complex with a full cellononaose ligand and a glycosyl-enzyme intermediate, that reveal details of the 'static' reaction coordinate. We also employ transition path sampling to determine the 'dynamic' reaction coordinate for the catalytic cycle. The glycosylation reaction coordinate contains components of forming and breaking bonds and a conformational change in the nucleophile. Deglycosylation proceeds via a product-assisted mechanism wherein the glycosylation product, cellobiose, positions a water molecule for nucleophilic attack on the anomeric carbon of the glycosyl-enzyme intermediate. In concert with previous structures, the present results reveal the complete hydrolytic reaction coordinate for this naturally and industrially important enzyme family.
糖苷水解酶(GHs)通过反转或保留机制切割碳水化合物中的糖苷键,后者通过两步机制进行,其中包括形成糖基-酶中间物。我们展示了 Hypocrea jecorina GH 家族 7 纤维二糖水解酶 Cel7A 的催化结构域的两个新结构,即与完整的纤维六糖配体和糖基-酶中间物的迈克尔加成复合物,揭示了“静态”反应坐标的细节。我们还采用过渡路径采样来确定催化循环的“动态”反应坐标。糖基化反应坐标包含形成和断裂键的成分以及亲核试剂的构象变化。去糖基化通过产物辅助机制进行,其中糖基化产物纤维二糖将水分子定位在糖基-酶中间物的糖苷碳原子上进行亲核攻击。与以前的结构相结合,本研究结果揭示了这种自然和工业上重要的酶家族的完整水解反应坐标。