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The Two-Species Model of transketolase explains donor substrate-binding, inhibition and heat-activation

We recently characterised a low-activity form of E. coli transketolase, TK(low), which also binds the cofactor thiamine pyrophosphate (TPP) with an affinity up to two-orders of magnitude lower than the previously known high TPP-affinity and high-activity form, TK(high), in the presence of Mg(2+). We...

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Detalles Bibliográficos
Autores principales: Wilkinson, Henry C., Dalby, Paul A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7057962/
https://www.ncbi.nlm.nih.gov/pubmed/32139871
http://dx.doi.org/10.1038/s41598-020-61175-z
Descripción
Sumario:We recently characterised a low-activity form of E. coli transketolase, TK(low), which also binds the cofactor thiamine pyrophosphate (TPP) with an affinity up to two-orders of magnitude lower than the previously known high TPP-affinity and high-activity form, TK(high), in the presence of Mg(2+). We observed previously that partial oxidation was responsible for increased TK(high) activity, while low-activity TK(low) was unmodified. In the present study, the fluorescence-based cofactor-binding assay was adapted to detect binding of the β-hydroxypyruvate (HPA) donor substrate to wild-type transketolase and a variant, S385Y/D469T/R520Q, that is active towards aromatic aldehydes. Transketolase HPA affinity again revealed the two distinct forms of transketolase at a TK(high):TK(low) ratio that matched those observed previously via TPP binding to each variant. The HPA dissociation constant of TK(low) was comparable to the substrate-inhibition dissociation constant, K(i)(HPA), determined previously. We provide evidence that K(i)(HPA) is a convolution of binding to the low-activity TK(low)-TK(low) dimer, and the TK(low) subunit of the partially-active TK(high)-TK(low) mixed dimer, where HPA binding to the TK(low) subunit of the mixed dimer results in inhibition of the active TK(high) subunit. Heat-activation of transketolase was similarly investigated and found to convert the TK(low) subunit of the mixed dimer to have TK(high)-like properties, but without oxidation.