Computational design of catalytic dyads and oxyanion holes for ester hydrolysis.

TitleComputational design of catalytic dyads and oxyanion holes for ester hydrolysis.
Publication TypeJournal Article
Year of Publication2012
AuthorsRichter, F., Blomberg R., Khare S. D., Kiss G., Kuzin A. P., Smith A. J. T., Gallaher J., Pianowski Z., Helgeson R. C., Grjasnow A., Xiao R., Seetharaman J., Su M., Vorobiev S., Lew S., Forouhar F., Kornhaber G. J., Hunt J. F., Montelione G. T., Tong L., Houk K. N., Hilvert D., & Baker D.
JournalJournal of the American Chemical Society
Volume134
Issue39
Pagination16197-206
Date Published2012 Oct 3
ISSN1520-5126
KeywordsPrimary Publication
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.

DOI10.1021/ja3037367
Custom1

http://www.ncbi.nlm.nih.gov/pubmed/22871159?dopt=Abstract

Alternate JournalJ. Am. Chem. Soc.