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Nucleophile Selectivity in the Acyl Transfer Reaction of a Designed Enzyme
Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry . Linköping University, The Institute of Technology.
Department of Chemistry, Biomedical Centre, Uppsala University, Uppsala, Sweden.
2005 (English)In: Biopolymers, Vol. 79, no 6, 292-299 p.Article in journal (Refereed) Published
Abstract [en]

The acyl transfer reaction of S-glutathionyl benzoate (GSB) is catalyzed by a rationally designed mutant of human glutathione transferase A1-1, A216H. The catalyzed reaction proceeds via the formation of an acyl intermediate and has been studied in the presence of nitrogen, oxygen, and sulfur nucleophiles to determine the selectivity with regards to nucleophile structure. Methanol was previously shown to react with the acyl intermediate and form the corresponding ester, methylbenzoate, under a significant rate enhancement. In the present investigation, the dependence on nucleophile structure and reactivity has been investigated. Ethane thiol gave rise to a larger rate enhancement in the enzyme-catalyzed reaction than ethanol, whereas ethylamine did not increase the reaction rate. The reactivities toward the acyl intermediate of primary and secondary alcohols with similar pKa values depended on the structure of the aliphatic chain, and 1-propanol was the most efficient alcohol. The reactivity of the oxygen nucleophiles was also found to depend strongly on pKa as 2,2,2-trifluoroethanol, with a pKa of 12.4, was the most efficient nucleophile of all that were tested. Saturation kinetics was observed in the case of 1-propanol, indicating a second binding site in the active site of A216H. The nucleophile selectivity of A216H provides the knowledge base needed for the further reengineering of A216H towards alternative substrate specificities.

Place, publisher, year, edition, pages
2005. Vol. 79, no 6, 292-299 p.
Keyword [en]
acyl transfer, enzyme catalysis, glutathione transferase, protein design, selectivity
National Category
Natural Sciences
URN: urn:nbn:se:liu:diva-13366DOI: 10.1002/bip.20351OAI: diva2:20489
Available from: 2005-09-23 Created: 2005-09-23
In thesis
1. Catalysis and Site-Specific Modification of Glutathione Transferases Enabled by Rational Design
Open this publication in new window or tab >>Catalysis and Site-Specific Modification of Glutathione Transferases Enabled by Rational Design
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes the rational design of a novel enzyme, a thiolester hydrolase, derived from human glutathione transferase (GST) A1-1 by the introduction of a single histidine residue. The first section of the thesis describes the design and the determination of the reaction mechanism. The design was based on the crystal structure of human GST A1-1 complexed with S-benzylglutathione. The resulting enzyme, A216H, catalyzed the hydrolysis of the non-natural substrate GSB, a thiolester of glutathione and benzoic acid. The reaction followed saturation kinetics with a kcat of 0.00078 min-1 and KM of 5 μM. The rate constant ratio, (kcat/KM)/kuncat, was found to be more than 107 M-1. The introduction of a single His residue in position 216 opened up a novel reaction pathway in human GST A1-1 and is a nice example of catalytic promiscuity. The substrate requirements were investigated and A216H was found to be selective since only two out of 18 GS-thiolesters tested were substrates for A216H. The reaction mechanism of the A216H-catalyzed hydrolysis of GSB was determined and found to proceed via an acyl intermediate at Y9. The hydrolysis was catalyzed by H216 that acts as a general base and the deacylation was found to be the rate-determining step. The Y9-intermediate could be selectively trapped by oxygen nucleophiles and primary alcohols, in particular 1-propanol and trifluoroethanol, were the most efficient. In addition, saturation kinetics was obtained in the acyl transfer reaction with 1-propanol indicating the presence of a second binding site in A216H.

The second section of this thesis describes the site-specific covalent modification of human GST A1-1. The addition of GSB to the wild-type protein results in a site-specific benzoylation of only one tyrosine residue, Y9, out of ten present in the protein (one out of totally 51 nucleophiles). The reaction was tested with five GST classes (Alpha, Mu, Pi, Theta and Omega) and found to be specific for the Alpha class isoenzymes. The covalent modification reaction was further refined to target a single lysine residue, K216, providing a more stable linkage in the form of an amide bond. The reaction was found to be versatile and approximately 50% of the GS-thiolesters tested acylated K216, including a fluorophore.

Place, publisher, year, edition, pages
Institutionen för fysik, kemi och biologi, 2005
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 964
Catalysis, Glutathione Transferases, Rational design, Protein design, Site-specific modification, thiolester hydrolysis
National Category
Biocatalysis and Enzyme Technology
urn:nbn:se:liu:diva-3962 (URN)91-85457-09-4 (ISBN)
Public defence
2005-09-30, 09:15 (English)
On the day of the public defence the status of article II was: Submitted and article IV was: In press.Available from: 2005-09-23 Created: 2005-09-23 Last updated: 2009-02-26

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