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 Mg²⁺. 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.