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 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.