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Ligand-Directed Labeling of a Single Lysine Residue in hGST A1-1 Mutants
Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry . Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry . Linköping University, The Institute of Technology.
2005 (English)In: Bioconjugate chemistry, ISSN 1043-1802 (print) 1520-4812 (online), Vol. 16, no 4, 1009-1018 p.Article in journal (Refereed) Published
Abstract [en]

Previously, we discovered that human glutathione transferase (hGST) A1-1 could be site-specifically acylated on a tyrosine residue (Y9) to form ester products using thiolesters of glutathione (GS-thiolesters) as acylating reagents. Out of a total of 20 GS-thiolester reagents tested, 15 (75%) are accepted by hGST A1-1 and thus this is a very versatile reaction. The present investigation was aimed at obtaining a more stable product, an amide bond, between the acyl group and the protein, in order to further increase the value of the reaction. Three lysine mutants (Y9K, A216K, and Y9F/A216K) were therefore prepared and screened against a panel of 18 GS-thiolesters. The Y9K mutant did not react with any of the reagents. The double mutant Y9F/A216K reacted with only one reagent, but in contrast, the A216K mutant could be acylated at the introduced lysine 216 with eight (44%) of the GS-thiolesters. The reaction can take place in the presence of glutathione and even in a crude cell lysate for five (28%) of the reagents. Through the screening process we obtained some basic rules relating to reagent requirements. We have thus produced a mutant (A216K) that can be rapidly and site-specifically modified at a lysine residue to form a stable amide linkage with a range of acyl groups. One of the successful reagents is a fluorophore that potentially can be used in downstream protein purification and protein fusion applications.

Place, publisher, year, edition, pages
2005. Vol. 16, no 4, 1009-1018 p.
Keyword [en]
human GST A1-1, site-specific covalent modification, tyrosine 9, alanine 216, lysine mutant, pre-programmed, ligand-directed protein modification, intramolecular acyl-transfer reaction
National Category
Natural Sciences
URN: urn:nbn:se:liu:diva-13367DOI: 10.1021/bc050111tOAI: diva2:20490
Available from: 2005-09-23 Created: 2005-09-23 Last updated: 2009-05-18
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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Hederos (Håkansson), SofiaKarlsson, BeatriceKerstin S., Broo
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