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Thiopurine S-methyltransferase - characterization of variants and ligand binding
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
2017 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Thiopurine S-methyltransferase (TPMT) belongs to the Class I S-adenosylmethionine-dependent methyltransferase (SAM-MT) super family of structurally related proteins. Common to the members of this large protein family is the catalysis of methylation reactions using S-adenosylmethionine (SAM) as a methyl group donor, although SAM-MTs act on a wide range of different substrates and carry out numerous biologically important functions. While the natural function of TPMT is unknown, this enzyme is involved in the metabolism of thiopurines, a class of pharmaceutical substances administered in treatment of immune-related disorders. Specifically, methylation by TPMT inactivates thiopurines and their metabolic intermediates, which reduces the efficacy of clinical treatment and increases the risk of adverse side effects. To further complicate matters, TPMT is a polymorphic enzyme with over 40 naturally occurring variants known to date, most of which exhibit lowered methylation activity towards thiopurines. Consequently, there are individual variations in TPMTmediated thiopurine inactivation, and the administered dose has to be adjusted prior to clinical treatment to avoid harmful side effects.

Although the clinical relevance of TPMT is well established, few studies have investigated the molecular causes of the reduced methylation activity of variant proteins. In this thesis, the results of biophysical characterization of two variant proteins, TPMT*6 (Y180F) and TPMT*8 (R215H), are presented. While the properties of TPMT*8 were indistinguishable from those of the wild-type protein, TPMT*6 was found to be somewhat destabilized. Interestingly, the TPMT*6 amino acid substitution did not affect the functionality or folding pattern of the variant protein. Therefore, the decreased in vivo functionality reported for TPMT*6 is probably caused by increased proteolytic degradation in response to the reduced stability of this protein variant, rather than loss of function.

Also presented herein are novel methodological approaches for studies of TPMT and its variants. Firstly, the advantages of using 8-anilinonaphthalene-1-sulfonic acid (ANS) to probe TPMT tertiary structure and active site integrity are presented. ANS binds exclusively to the native state of TPMT with high affinity (KD ~ 0.2 μm) and a 1:1 ratio. The stability of TPMT was dramatically increased by binding of ANS, which was shown to co-localize with the structurally similar adenine moiety of the cofactor SAM. Secondly, an enzyme activity assay based on isothermal titration calorimetry (ITC) is presented. Using this approach, the kinetics of 6-MP and 6-TG methylation by TPMT has been characterized.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2017. , 50 p.
Series
Linköping Studies in Science and Technology. Thesis, ISSN 0280-7971 ; 1776
Keyword [en]
Thiopurine S-methyltransferase (TPMT); protein stability; enzyme kinetics; ligand binding; isothermal titration calorimetry (ITC); 8-anilinonaphthalene-1-sulfonic acid (ANS)
National Category
Chemical Sciences Biochemistry and Molecular Biology Medicinal Chemistry Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biocatalysis and Enzyme Technology Structural Biology
Identifiers
URN: urn:nbn:se:liu:diva-136558ISBN: 9789176855300 (print)OAI: oai:DiVA.org:liu-136558DiVA: diva2:1089519
Presentation
2017-05-16, Planck, Fysihuset, Campus Valla, Linköpings universitet, Linköping, 14:00 (Swedish)
Opponent
Supervisors
Available from: 2017-04-20 Created: 2017-04-20 Last updated: 2017-05-10Bibliographically approved

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