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

Henry C Wilkinson,Paul A Dalby

doi:10.1038/s41598-020-61175-z

Abstract

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.

Highlights

We recently characterised a low-activity form of E. coli transketolase, TKlow, which 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 examine directly the binding of substrate to the transketolase species subpopulations for both wild type and the variant S385Y/D469T/R520Q, and provide further evidence that transketolase is a redox-regulated enzyme, that could potentially play a major role in the control of flux through the phosphate pathway (PPP) during oxidative stress
We report the TPP-binding parameters for S385Y/D469T/R520Q, a variant that was previously engineered for activity towards aromatic aldehydes[26]

Summary

Introduction

We recently characterised a low-activity form of E. coli transketolase, TKlow, which 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+. Transketolase displays ping-pong kinetics and catalyses two sequential half-reactions; formation of the dihydroxyethyl-TPP (DHE-TPP) carbanion intermediate, followed by transfer of this two-carbon ketol group from the carbanion intermediate to an acceptor aldehyde, returning the enzyme to its starting state. It is Department of Biochemical Engineering, University College London, London, WC1E 6BT, UK. Www.nature.com/scientificreports thought that there is considerable communication between active sites in order to coordinate the ping-pong kinetics between active sites, such that catalysis alternates between active sites, giving rise to the ‘half-of-the-sites reactivity’ phenomenon that has been observed in transketolase[7] and other TPP-dependent enzymes[8,9,10,11]

Methods

Results

Conclusion