Schutt Timothy C, Bharadwaj Vivek S, Granum David M, Maupin C Mark
Chemical and Biological Engineering Department, Colorado School of Mines, 1500 Illinois Street, Golden, Colorado 80401, USA.
Phys Chem Chem Phys. 2015 Jul 14;17(26):16947-58. doi: 10.1039/c5cp01801c.
The ability to utilize biomass as a feedstock for liquid fuel and value-added chemicals is dependent on the efficient and economic utilization of lignin, hemicellulose, and cellulose. In current bioreactors, cellulases are used to convert crystalline and amorphous cellulose to smaller oligomers and eventually glucose by means of cellulase enzymes. A critical component of the enzyme catalyzed hydrolysis reaction is the degree to which the enzyme can facilitate substrate ring deformation from the chair to a more catalytically active conformation (e.g. skewed boat) at the -1 subsite. Presented here is an evaluation of the impact of the protonation state for critical active site residues (i.e. Glu212, Asp214, Glu217, and His228) in Melanocarpus albomyces (Ma) Cellobiohydrolase Cel7B on the substrate's orientation and ring conformation. It is found that the protonation state of the active site can disrupt the intra-enzyme hydrogen bonding network and enhance the sampling of various ring puckering conformations for the substrate ring at the +1 and -1 subsites. In particular it is observed that the protonation state of Asp214 dictates the accessibility of the glycosidic bond to the catalytic acid/base Glu217 by influencing the φ/ψ dihedral angles and the puckering of the ring structure. The protonation-orientation-conformation analysis has revealed an active site that primarily utilizes two highly coupled protonation schemes; one protonation scheme to orient the substrate and generate catalytically favorable substrate geometries and ring puckering conformations and another protonation scheme to hydrolyze the glycosidic bond. In addition to identifying how enzymes utilize protonation state to manipulate substrate geometry, this study identifies possible directions for improving catalytic activity through protein engineering.
将生物质用作液体燃料和增值化学品的原料的能力取决于木质素、半纤维素和纤维素的高效经济利用。在当前的生物反应器中，纤维素酶用于通过纤维素酶将结晶和无定形纤维素转化为较小的低聚物并最终转化为葡萄糖。酶催化水解反应的一个关键组成部分是酶能够促进底物环在 -1 亚位点从椅式构象变形为更具催化活性的构象（例如扭曲船式）的程度。本文介绍了对黑曲霉（Ma）纤维二糖水解酶Cel7B中关键活性位点残基（即Glu212、Asp214、Glu217和His228）的质子化状态对底物取向和环构象的影响的评估。发现活性位点的质子化状态可以破坏酶内氢键网络，并增强底物环在 +1 和 -1 亚位点的各种环皱缩构象的采样。特别观察到，Asp214的质子化状态通过影响φ/ψ二面角和环结构的皱缩来决定糖苷键对催化酸/碱Glu217的可及性。质子化 - 取向 - 构象分析揭示了一个主要利用两种高度耦合的质子化方案的活性位点；一种质子化方案用于使底物取向并产生催化有利的底物几何形状和环皱缩构象，另一种质子化方案用于水解糖苷键。除了确定酶如何利用质子化状态来操纵底物几何形状外，本研究还确定了通过蛋白质工程提高催化活性的可能方向。
