We recently characterised a low-activity form of E. coli transketolase, TKlow, 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, TKhigh, in the presence of Mg2+. We observed previously that partial oxidation was responsible for increased TKhigh activity, while low-activity TKlow 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 TKhigh:TKlow ratio that matched those observed previously via TPP binding to each variant. The HPA dissociation constant of TKlow was comparable to the substrate-inhibition dissociation constant, KiHPA, determined previously. We provide evidence that KiHPA is a convolution of binding to the low-activity TKlow-TKlow dimer, and the TKlow subunit of the partially-active TKhigh-TKlow mixed dimer, where HPA binding to the TKlow subunit of the mixed dimer results in inhibition of the active TKhigh subunit. Heat-activation of transketolase was similarly investigated and found to convert the TKlow subunit of the mixed dimer to have TKhigh-like properties, but without oxidation.