Abstract
Transketolase (TK) cofactor binding has been studied extensively over many years, yet certain mysteries remain, such as a lack of consensus on the cooperativity of thiamine pyrophosphate (TPP) binding into the two active sites, in the presence and absence of the divalent cation, Mg2+. Using a novel fluorescence-based assay, we determined directly the dissociation constants and cooperativity of TPP binding and provide the first comprehensive study over a broad range of cofactor concentrations. We confirmed the high-affinity dissociation constants and revealed a dependence of both the affinity and cooperativity of binding on [Mg2+], which explained the previous lack of consensus. A second, discrete and previously uncharacterised low-affinity TPP binding-site was also observed, and hence indicated the existence of two forms of TK with high- (TKhigh) and low-affinity (TKlow). The relative proportions of each dimer were independent of the monomer-dimer transition, as probed by analytical ultracentrifugation at various [TK]. Mass spectrometry revealed that chemical oxidation of TKlow led to the formation of TKhigh, which was 22-fold more active than TKlow. Finally, we propose a two-species model of transketolase activation that describes the interconversions between apo-/holo-TKhigh and TKlow, and the potential to significantly improve biocatalytic activity by populating only the most active form.
Highlights
Transketolase (TK) cofactor binding has been studied extensively over many years, yet certain mysteries remain, such as a lack of consensus on the cooperativity of thiamine pyrophosphate (TPP) binding into the two active sites, in the presence and absence of the divalent cation, Mg2+
The signal generated was corrected for the inner filter effect (IFE), that arises from strong absorption of a proportion of the incident light by free ligand (TPP) before it can excite the sample (‘primary’ IFE), and which decreases the observed fluorescence
It is unclear why TKlow was not detected in other cofactor-binding studies, but this could have been one, some or all of the following reasons: (a) the larger range of cofactor concentrations used in this study; (b) the larger datasets taken at each [Mg2+]; (c) there may be no inducible absorption band at 320 nm upon cofactor binding to TKlow; and (d) TKlow activity is negligible relative to TK with high- (TKhigh) and so invisible to measurements of cofactor affinity based on activity measurements
Summary
Transketolase (TK) cofactor binding has been studied extensively over many years, yet certain mysteries remain, such as a lack of consensus on the cooperativity of thiamine pyrophosphate (TPP) binding into the two active sites, in the presence and absence of the divalent cation, Mg2+. Yeast and E. coli apo-transketolases exist as monomers that form homodimers at higher protein concentrations[7] Upon cofactor binding, both the apo-monomer and apo-dimer form a catalytically active homodimer of apparently structurally-identical subunits, with two active sites per homodimer located at the subunit interface (Fig. 1)[8,9]. Cofactor binding to the inactive apo-transketolase dimer leads to the structural organisation of two disordered cofactor-binding loops in the active site, to form the active holo-TK homodimer[11]. Following the unexpected detection of two distinct transketolase species with stark differences in affinity and activity, the origin of the ratio of the two species was probed further, and linked to a specific chemical oxidation, namely single- and double-oxidation of an active-site Cys[157] thiol (RSH), to form a sulfenic acid (RSOH) or sulfinic acid (RSO2H), respectively
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