Title: Computational design of catalytic dyads and oxyanion holes for ester hydrolysis
Authors : Richter, Florian
Blomberg, Rebecca
Khare, Sagar D.
Kiss, Gert
Kuzin, Alexandre P.
Smith, Adam J. T.
Gallaher, Jasmine
Pianowski, Zbigniew
Helgeson, Roger C.
Grjasnow, Alexej
Xiao, Rong
Seetharaman, Jayaraman
Su, Min
Vorobiev, Sergey
Lew, Scott
Forouhar, Farhad
Kornhaber, Gregory J.
Hunt, John F.
Montelione, Gaetano T.
Tong, Liang
Houk, K. N.
Hilvert, Donald
Baker, David
Published in : Journal of the American Chemical Society
Volume(Issue) : 134
Issue : 39
Pages : 16197
Pages to: 16206
Publisher / Ed. Institution : American Chemical Society
Issue Date: 2012
License (according to publishing contract) : Licence according to publishing contract
Type of review: Peer review (Publication)
Language : English
Subjects : Catalytic domain; Esterases; Esters; Hydrogen bonding; Hydrolysis; Kinetics; Biocatalysis; Drug design; Molecular models
Subject (DDC) : 540: Chemistry
Abstract: 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 (k(cat)/K(M)) of 400 M(-1) s(-1) 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.
Departement: Life Sciences and Facility Management
Publication type: Article in scientific Journal
DOI : 10.1021/ja3037367
ISSN: 0002-7863
1520-5126
URI: https://digitalcollection.zhaw.ch/handle/11475/9665
Appears in Collections:Publikationen Life Sciences und Facility Management

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