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Novel insights into transketolase activation by cofactor binding identifies two native species subpopulations

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. Green open access

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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.

Type: Article
Title: Novel insights into transketolase activation by cofactor binding identifies two native species subpopulations
Location: England
Open access status: An open access version is available from UCL Discovery
DOI: 10.1038/s41598-019-52647-y
Publisher version: https://doi.org/10.1038/s41598-019-52647-y
Language: English
Additional information: 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/.
UCL classification: UCL
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Chemical Engineering
URI: https://discovery.ucl.ac.uk/id/eprint/10085810
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