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
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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.
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