Thiopurine S-methyltransferase - characterization of variants and ligand binding

• 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.

Ämnesord

NATURVETENSKAP  -- Kemi (hsv//swe)

NATURAL SCIENCES  -- Chemical Sciences (hsv//eng)

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinska och farmaceutiska grundvetenskaper -- Läkemedelskemi (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Basic Medicine -- Medicinal Chemistry (hsv//eng)

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinsk bioteknologi -- Medicinsk bioteknologi (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Medical Biotechnology -- Medical Biotechnology (hsv//eng)

TEKNIK OCH TEKNOLOGIER  -- Industriell bioteknik -- Biokatalys och enzymteknik (hsv//swe)

ENGINEERING AND TECHNOLOGY  -- Industrial Biotechnology -- Biocatalysis and Enzyme Technology (hsv//eng)

NATURVETENSKAP  -- Biologi -- Strukturbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Structural Biology (hsv//eng)

Nyckelord

Thiopurine S-methyltransferase (TPMT); protein stability; enzyme kinetics; ligand binding; isothermal titration calorimetry (ITC); 8-anilinonaphthalene-1-sulfonic acid (ANS)

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