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Importance of a hypervariable active-site residue in human Mu class glutathione transferases catalyzing the bioactivation of chemotherapeutic thiopurine prodrugs
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
2007 (English)In: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1770, no 8, 1098-1103 p.Article in journal (Refereed) Published
Abstract [en]

Glutathione transferases (GSTs) catalyze the bioactivation of the thiopurine prodrugs azathioprine, cis-6-(2-acetylvinylthio)purine (cAVTP) and trans-6-(2-acetylvinylthio)guanine (tAVTG), thereby releasing the antimetabolites 6-mercaptopurine and 6-thioguanine. In the GST Mu class, GST M1-1 has the highest catalytic efficiency, whereas GST M2-2 and other enzymes are less active. In the evolution of Mu class GSTs, residue 210 appears hypervariable and has particular functional significance. We demonstrate that the catalytic activity of GST M1-1 with cAVTP or tAVTG is successively diminished when wild-type Ser-210 is mutated into Ala followed by Thr. Conversely, mutating wild-type Thr-210 in GST M2-2 into Ala and Ser enhanced the corresponding activities. Comparisons were also made with GST M2-2 distinguished by Gly or Pro in position 210, as well as wild-type GSTs M4-4 and M5-5. The results suggest that the hydroxyl group of Ser in position 210 stabilizes the transition state of the GST-catalyzed reaction. The low activity of GSTs containing Thr in position 210 is probably due to steric hindrance caused by the β-methyl group of the side chain. The ratios of the different catalytic efficiencies were translated into differences in the Gibbs free energies of transition state stabilization. The effects of the mutations were qualitatively parallel for the alternative substrates, but vary significantly in magnitude. From the evolutionary perspective the data show that a point mutation can alternatively enhance or attenuate the activity with a particular substrate and illustrate the functional plasticity of GSTs.

Place, publisher, year, edition, pages
2007. Vol. 1770, no 8, 1098-1103 p.
Keyword [en]
Glutathione transferase, Thiopurine prodrugs, Functional plasticity, Active-site mutation, Modulated activity
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-94759DOI: 10.1016/j.bbagen.2007.04.001ISI: 000248501000003PubMedID: 17493759OAI: oai:DiVA.org:uu-94759DiVA: diva2:168730
Available from: 2006-08-31 Created: 2006-08-31 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Role of Multiple Glutathione Transferases in Bioactivation of Thiopurine Prodrugs: Studies of Human Soluble Glutathione Transferases from Alpha, Kappa, Mu, Omega, Pi, Theta, and Zeta Classes
Open this publication in new window or tab >>Role of Multiple Glutathione Transferases in Bioactivation of Thiopurine Prodrugs: Studies of Human Soluble Glutathione Transferases from Alpha, Kappa, Mu, Omega, Pi, Theta, and Zeta Classes
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A screening method was developed for identification of catalytically active enzymes in combinatorial cDNA libraries of mutated glutathione transferase (GST) derivatives expressed in E. coli. The method is based on spraying monochlorobimane (MCB) directly over bacterial colonies growing on agar. The substrate MCB become fluorescent under UV light, when the bacterial colony contains active GSTs catalyzing the conjugation with endogenous glutathione. Eleven out of twelve GSTs investigated where active with MCB. This method can be used to screen libraries generated from most cytosolic GSTs in the search for proteins with altered functions and structures. Azathioprine (Aza), a thiopurine that has been used clinically for 40 years was investigated with 14 GSTs. Three enzymes showed prominent catalytic activities with Aza and all of them are highly expressed in the liver. We estimated the contribution of the three enzymes GSTs A1-1, A2-2 and M1-1 bioactivation of Aza in the liver and concluded that it was about 2 orders of magnitude more effective than the uncatalyzed reaction. GST bioactivation of Aza could clarify aspects of idiosyncratic reactions observed in some individuals. Two other thiopurine prodrugs, cis-acetylvinylthiopurine (cAVTP) and trans-acetylvinylthioguanine (tAVTG), were investigated with the same 14 GSTs. The results displayed diverse catalytic activities. A mechanism of consecutive reactions was proposed. The studies contribute to knowledge under what conditions the drug should optimally be administered. A study of the same prodrugs with several mutants from the Mu class characterized by a point mutation of a hypervarible residue. We conclude that the effects of the mutations were qualitatively parallel for cAVTP and tAVTG, but they vary significantly in magnitude; steric hindrance may interfere with transition-state stabilization. From the evolutionary perspective the data show that a point mutation can alternatively enhance or attenuate the activity with a particular substrate and illustrate the functional plasticity of GSTs.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 47 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 208
Keyword
Biochemistry, Glutathione Transferases, Thiopurines, Prodrug, Bioactivation, Screening Method, Chemotherapy, Functional plasticity, Modulated activity, Biokemi
Identifiers
urn:nbn:se:uu:diva-7102 (URN)91-554-6630-3 (ISBN)
Public defence
2006-09-22, B21, BMC, Husarg 3, Uppsala, 10:15
Opponent
Supervisors
Available from: 2006-08-31 Created: 2006-08-31 Last updated: 2011-07-18Bibliographically approved

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