We recently characterised a low-activity form of E. coli transketolase, TK , 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 , in the presence of Mg . We observed previously that partial oxidation was responsible for increased TK activity, while low-activity TK 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 :TK ratio that matched those observed previously via TPP binding to each variant. The HPA dissociation constant of TK was comparable to the substrate-inhibition dissociation constant, K , determined previously. We provide evidence that K is a convolution of binding to the low-activity TK -TK dimer, and the TK subunit of the partially-active TK -TK mixed dimer, where HPA binding to the TK subunit of the mixed dimer results in inhibition of the active TK subunit. Heat-activation of transketolase was similarly investigated and found to convert the TK subunit of the mixed dimer to have TK -like properties, but without oxidation.