eprintid: 10085810 rev_number: 20 eprint_status: archive userid: 608 dir: disk0/10/08/58/10 datestamp: 2019-11-13 10:41:23 lastmod: 2021-10-16 22:49:12 status_changed: 2019-11-13 10:41:23 type: article metadata_visibility: show creators_name: Wilkinson, HC creators_name: Dalby, PA title: Novel insights into transketolase activation by cofactor binding identifies two native species subpopulations ispublished: pub divisions: UCL divisions: B04 divisions: C05 divisions: F47 divisions: F43 note: This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. abstract: Transketolase (TK) cofactor binding has been studied extensively over many years, yet certain mysteries remain, such as a lack of consensus on the cooperativity of thiamine pyrophosphate (TPP) binding into the two active sites, in the presence and absence of the divalent cation, Mg2+. Using a novel fluorescence-based assay, we determined directly the dissociation constants and cooperativity of TPP binding and provide the first comprehensive study over a broad range of cofactor concentrations. We confirmed the high-affinity dissociation constants and revealed a dependence of both the affinity and cooperativity of binding on [Mg2+], which explained the previous lack of consensus. A second, discrete and previously uncharacterised low-affinity TPP binding-site was also observed, and hence indicated the existence of two forms of TK with high- (TKhigh) and low-affinity (TKlow). The relative proportions of each dimer were independent of the monomer-dimer transition, as probed by analytical ultracentrifugation at various [TK]. Mass spectrometry revealed that chemical oxidation of TKlow led to the formation of TKhigh, which was 22-fold more active than TKlow. Finally, we propose a two-species model of transketolase activation that describes the interconversions between apo-/holo-TKhigh and TKlow, and the potential to significantly improve biocatalytic activity by populating only the most active form. date: 2019-11-06 date_type: published official_url: https://doi.org/10.1038/s41598-019-52647-y oa_status: green full_text_type: pub language: eng primo: open primo_central: open_green verified: verified_manual elements_id: 1717493 doi: 10.1038/s41598-019-52647-y pii: 10.1038/s41598-019-52647-y lyricists_name: Dalby, Paul lyricists_name: Wilkinson, Henry lyricists_id: PADAL59 lyricists_id: HCWIL75 actors_name: Kalinowski, Damian actors_id: DKALI47 actors_role: owner full_text_status: public publication: Scientific Reports volume: 9 article_number: 16116 event_location: England issn: 2045-2322 citation: Wilkinson, HC; Dalby, PA; (2019) Novel insights into transketolase activation by cofactor binding identifies two native species subpopulations. Scientific Reports , 9 , Article 16116. 10.1038/s41598-019-52647-y <https://doi.org/10.1038/s41598-019-52647-y>. Green open access document_url: https://discovery.ucl.ac.uk/id/eprint/10085810/1/Dalby_Novel%20insights%20into%20transketolase%20activation%20by%20cofactor%20binding%20identifies%20two%20native%20species%20subpopulations_VoR.pdf