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Downsizing of Enzymes by Chemical Methods: Arginine Mimics with Low pK(a) Values Increase the Rates of Hydrolysis of RNA Model Compounds
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
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2009 (English)In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 48, no 36, 6722-6725 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2009. Vol. 48, no 36, 6722-6725 p.
Keyword [en]
de novo design, homogeneous catalysis, hydrolysis, peptides, phosphodiesters
National Category
Chemical Sciences
URN: urn:nbn:se:uu:diva-100486DOI: 10.1002/anie.200900595ISI: 000269361300034PubMedID: 19655357OAI: oai:DiVA.org:uu-100486DiVA: diva2:210357
Available from: 2009-04-01 Created: 2009-04-01 Last updated: 2010-10-26Bibliographically approved
In thesis
1. Expanding the Amino Acid Alphabet by Design: Enhanced and Controlled Catalytic Activity in Folded Polypeptide Catalysts
Open this publication in new window or tab >>Expanding the Amino Acid Alphabet by Design: Enhanced and Controlled Catalytic Activity in Folded Polypeptide Catalysts
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis addresses structure and reactivity of polypeptide catalysts in reactions that mimic the hydrolysis of RNA and DNA. A designed helix-loop-helix motif was used as a scaffold where the amino acid residues were systematically varied. The reactivity of a previously reported catalyst, HNI, was evaluated and the catalytic residues of the active site, Arg and His, were replaced in a stepwise manner by the artificial amino acid Gcp. Gcp has a guanidinocarbonyl pyrrole side chain, i.e. a side chain that mimics that of Arg but with a lower pKa. Gcp was used to replace both histidine and arginine in the polypeptide catalysts and was able to bind phosphate as well as carry out general base catalysis.

The parent polypeptide HNI was shown to catalyse phosphoryl transfer reactions of phosphodiesters in an active site with two His and two Arg residues. The performance of the active site was improved by the introduction of two Tyr residues to form the catalyst HJ1 designed to provide nucleophilic catalysis in the hydrolysis of DNA model substrates.

To improve the catalytic activity beyond that of HJI, Gcp was introduced to replace Arg and His residues in the HN1 scaffold. The designed catalyst JL3 was capable of a 150-fold rate enhancement compared to HNI in the reaction of the substrate HPNP, representing the first step in RNA hydrolysis. Mechanistic studies of JL3 catalysis suggested that the pKa value of the Gcp residue in the folded polypeptides was around 5. In combination with the observation of a solvent kinetic isotope effect of 1.7 the Gcp residue was proposed to provide general base catalysis and transition state stabilisation in the reaction of uridine 3′-2,2,2-trichloroethylphosphate, a realistic RNA model with a leaving group pKa of 12.5. The JL3 polypeptide catalyst followed saturation kinetics with a kcat/KM of 1.08 x 10-3 M-1s-1.

The introduction of a designed photoswitchable amino acid in a catalytic polypeptide allowed the activity of the polypeptide in the reaction with an activated ester to be under photochemical control. Photoisomerization of this switch altered the structure of the polypeptide and affected the catalytic activity of the polypeptide catalyst.

The chemical synthesis of designed molecules expands the amino acid alphabet and makes it possible to downsize enzymatic functions. It opens up possibilities for the production of novel biocatalysts that can catalyse natural as well as non-natural reactions.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 62 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 630
National Category
Other Basic Medicine
Research subject
Organic Chemistry
urn:nbn:se:uu:diva-100324 (URN)978-91-554-7485-0 (ISBN)
Public defence
2009-05-08, B42, BMC, Husargatan 3, Uppsala, 10:15 (English)
Available from: 2009-04-17 Created: 2009-03-30 Last updated: 2009-04-20Bibliographically approved

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Lindgren, N. Johan V.
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