Witkowska, D, Cox, HL, Hall, TC et al. (3 more authors) (2018) Analysis of substrate binding in individual active sites of bifunctional human ATIC. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 1866 (2). pp. 254-263. ISSN 1570-9639
Abstract
Aminoimidazolecarboxamide ribonucleotide formyl transferase (AICARFT): Inosine monophosphate cyclohydrolase (IMPCH, collectively called ATIC) is a bifunctional enzyme that catalyses the penultimate and final steps in the purine de novo biosynthesis pathway. The bifunctional protein is dimeric and each monomer contains two different active sites both of which are capable of binding nucleotide substrates, this means to a potential total of four distinct binding events might be observed. Within this work we used a combination of site-directed and truncation mutants of ATIC to independently investigate the binding at these two sites using calorimetry. A single S10W mutation is sufficient to block the IMPCH active site allowing investigation of the effects of mutation on ligand binding in the AICARFT active site. The majority of nucleotide ligands bind selectively at one of the two active sites with the exception of xanthosine monophosphate, XMP, which, in addition to binding in both AICARFT and IMPCH active sites, shows evidence for cooperative binding with communication between symmetrically-related active sites in the two IMPCH domains. The AICARFT site is capable of independently binding both nucleotide and folate substrates with high affinity however no evidence for positive cooperativity in binding could be detected using the model ligands employed in this study.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2017 Elsevier B.V. This is an author produced version of a paper published in Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. Uploaded in accordance with the publisher's self-archiving policy. |
Keywords: | Bifunctional enzymes; Isothermal titration calorimetry; Multiple binding sites |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Biology (Leeds) The University of Leeds > Faculty of Engineering & Physical Sciences (Leeds) > School of Chemistry (Leeds) > Organic Chemistry (Leeds) |
Depositing User: | Symplectic Publications |
Date Deposited: | 17 Oct 2017 14:05 |
Last Modified: | 16 Oct 2018 00:38 |
Status: | Published |
Publisher: | Elsevier |
Identification Number: | 10.1016/j.bbapap.2017.10.005 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:122681 |