Computational Design of Catalytic Dyads and Oxyanion Holes for Ester Hydrolysis
详细信息 查看全文
- 作者：Florian Richter ; Rebecca Blomberg ; Sagar D. Khare ; Gert Kiss ; Alexandre P. Kuzin ; Adam J. T. Smith ; Jasmine Gallaher ; Zbigniew Pianowski ; Roger C. Helgeson ; Alexej Grjasnow ; Rong Xiao ; Jayaraman Seetharaman ; Min Su ; Sergey Vorobiev ; Scott Lew ; Farhad Forouhar ; Gregory J. Kornhaber ; John F. Hunt ; Gaetano T. Montelione ; Liang Tong ; K. N. Houk ; Donald Hilvert ; David Baker
- 刊名：The Journal of the American Chemical Society
- 出版年：2012
- 出版时间：October 3, 2012
- 年：2012
- 卷：134
- 期：39
- 页码：16197-16206
- 全文大小：446K
- 年卷期：v.134,no.39(October 3, 2012)
- ISSN：1520-5126
文摘
Nucleophilic catalysis is a general strategy for accelerating ester and amide hydrolysis. In natural active sites, nucleophilic elements such as catalytic dyads and triads are usually paired with oxyanion holes for substrate activation, but it is difficult to parse out the independent contributions of these elements or to understand how they emerged in the course of evolution. Here we explore the minimal requirements for esterase activity by computationally designing artificial catalysts using catalytic dyads and oxyanion holes. We found much higher success rates using designed oxyanion holes formed by backbone NH groups rather than by side chains or bridging water molecules and obtained four active designs in different scaffolds by combining this motif with a Cys-His dyad. Following active site optimization, the most active of the variants exhibited a catalytic efficiency (kcat/KM) of 400 M鈥? s鈥? for the cleavage of a p-nitrophenyl ester. Kinetic experiments indicate that the active site cysteines are rapidly acylated as programmed by design, but the subsequent slow hydrolysis of the acyl-enzyme intermediate limits overall catalytic efficiency. Moreover, the Cys-His dyads are not properly formed in crystal structures of the designed enzymes. These results highlight the challenges that computational design must overcome to achieve high levels of activity.