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标题：Novel insights into transketolase activation by cofactor binding identifies two native species subpopulations
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作者：Henry C. Wilkinson ; Paul A. Dalby
期刊名称：Scientific Reports
电子版ISSN：2045-2322
出版年度：2019
卷号：9
期号：1
页码：1-13
DOI：10.1038/s41598-019-52647-y
出版社：Springer Nature
摘要：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, Mg 2+ . 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 [Mg 2+ ], 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- (TK high ) and low-affinity (TK low ). 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 TK low led to the formation of TK high , which was 22-fold more active than TK low . Finally, we propose a two-species model of transketolase activation that describes the interconversions between apo-/holo-TK high and TK low , and the potential to significantly improve biocatalytic activity by populating only the most active form.