Rungrotmongkol Thanyada, Decha Panita, Sompornpisut Pornthep, Malaisree Maturos, Intharathep Pathumwadee, Nunthaboot Nadtanet, Udommaneethanakit Thanyarat, Aruksakunwong Ornjira, Hannongbua Supot
Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
Proteins. 2009 Jul;76(1):62-71. doi: 10.1002/prot.22318.
Combined quantum mechanical/molecular mechanical (QM/MM) techniques have been applied to investigate the detailed reaction mechanism of the first step of the acylation process by furin in which the cleavage site of the highly pathogenic avian influenza virus subtype H5N1 (HPH5) acts as its substrate. The energy profile shows a simultaneous mechanism, known as a concerted reaction, of the two subprocesses: the proton transfer from Ser368 to His194 and the nucleophilic attack on the carbonyl carbon of the scissile peptide of the HPH5 cleavage site with a formation of tetrahedral intermediate (INT). The calculated energy barrier for this reaction is 16.2 kcal.mol(-1) at QM/MM B3LYP/6-31+G*//PM3-CHARMM22 level of theory. Once the reaction proceeds, the ordering of the electrostatic stabilization by protein environment is of the enzyme-substrate < transition state < INT complexes. Asp153 was found to play the most important role in the enzymatic reaction by providing the highest degree of intermediate complex stabilization. In addition, the negatively charged carbonyl oxygen of INT is well stabilized by the oxyanion hole constructed by Asn295's carboxamide and Ser368's backbone.
量子力学/分子力学（QM/MM）联合技术已被用于研究弗林蛋白酶酰化过程第一步的详细反应机制，其中高致病性甲型流感病毒H5N1亚型（HPH5）的裂解位点作为其底物。能量分布图显示了两个子过程的同步机制，即所谓的协同反应：质子从Ser368转移到His194，以及对HPH5裂解位点可裂解肽羰基碳的亲核攻击，形成四面体中间体（INT）。在QM/MM B3LYP/6-31+G*//PM3-CHARMM22理论水平下，该反应的计算能垒为16.2 kcal·mol⁻¹。一旦反应进行，蛋白质环境的静电稳定顺序为酶-底物<过渡态<INT复合物。发现Asp153通过提供最高程度的中间体复合物稳定作用，在酶促反应中发挥最重要作用。此外，INT带负电荷的羰基氧通过由Asn295的羧酰胺和Ser368的主链构成的氧负离子洞得到很好的稳定。
