Novel insights into transketolase activation by cofactor binding identifies two native species subpopulations

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.


Section 3: Comparison of experimental TPP-binding data fitted to a single-or double-Hill function
The double-Hill function: = where q is the fractional saturation, the fraction of [protein] that is bound to ligand; B max(high) is the proportion of TPP that binds to TK high ; a.B max(high) is the proportion of TPP that binds to TK low ; [L] is the ligand (TPP) concentration; n high and n low are the Hill coefficients of TK high and TK low , respectively; and K d(high) and K d(low) are the dissociation constants of TK high and TK low , respectively.

Figure S3
: Experimental data of 0.05 mg/mL wild-type transketolase binding to TPP at a) and b) 0 mM; c) and d) 1 mM; e) and f) 4.5 mM; G) and H) 9 mM; and I) and J) 18 mM Mg 2+ . Experimental data-points were fitted to either a single-(black) or double-(black) Hill function. The data is presented on a logarithmic x-axis to demonstrate the superior fit to the double-Hill function.
The TPP-binding parameters determined using the single Hill (Table S1) have no relatability to previously determined dissociation constants and Hill coefficients; at higher [Mg 2+ ], the fits either had very large associated errors or the data couldn't be fitted to the single-Hill function. However, those of TK high , determined by fitting the data to the double-Hill function (Table S2), correlated well with previously reported values. We therefore conclude that we have detected two independent binding events, both cooperative, which fitted best to a double-Hill function.   6

Section 5: Lysis and purification in the presence of 10 mM β-mercaptoethanol does not impact
%B max(high) Figure S5: The mass spectra of wild-type transketolase expressed in the absence of β-mercaptoethanol and cumene hydroperoxide, then lysed and purified in the a) absence and b) presence of 10 mM βmercaptoethanol. The major peak (green) corresponds to unmodified transketolase (TK low ), while the next two peaks (blue and cyan) correspond to modified transketolase (collectively TK high ). Higher molecularweight peaks correspond to over-oxidised TK, which are likely inactive.

Section 6: Ruling out alternative explanations for the observed %B max(high) & TPP-binding a) Allostery
Allosteric-activator binding of TPP to transketolase is unlikely given the importance of TPP to the reaction mechanism, and the scarcity of TPP in the cell, and there is no kinetic or structural evidence of such binding outside of the two active-sites. It is therefore likely that the low-affinity binding site is located within the same active-site binding pocket as the high-affinity binding site. Indeed, all X-ray crystal structures of holo-transketolase showed only two TPP molecules per dimer 2,3 .

b) Asymmetric TPP binding to non-identical active sites in all homodimers
The presence of two TPP-binding events with significantly different affinities may at first glance support the hypothesis that two TPP molecules bind the two binding sites of a single transketolase dimer asymmetrically; one with high and one with low affinity. This notion is dispelled by several observations.
Firstly, we have determined that two independent binding events occur, both cooperative in nature. The observed binding events must therefore be more than cooperativity between only two sites. Furthermore, the dissociation constant of the low-affinity binding site detected in this study is 42-fold and 600-fold greater than those of the two non-equivalent, negatively cooperative active sites reported previously 4 , suggesting the low-affinity TPP binding site reported here is an entirely different binding site. Indeed, early work on transketolase cofactor binding detected an additional binding site with a significantly higher K d than the two non-equivalent 'high-affinity' binding sites detected, but no dissociation constant was obtained, nor was it characterised 5 . The dissociation constant of the high affinity site reported here was between those of the two non-equivalent sites reported previously 4 , as would be expected if two dissociation constants are analysed as a single dissociation constant using the Hill function.

c) Formation of an intermediate state during TPP binding
Transketolase cofactor-binding and activation has been shown to occur in at least two general steps.
The first stage is fast and readily reversible and results in the formation of the catalytically inactive TK---TPP primary complex. Subsequent conformational changes convert the primary complex into catalytically active holo-transketolase, TK*-TPP. The second step is quasi-irreversible and slow in nature 5,6 . In theory, it is possible that TK high and TK low may be synonymous with TK---TPP and TK*-TPP, and that the TK low population is unable to undergo the second, quasi-irreversible step to TK high . However, the maximum TPP concentration used in the previous kinetic experiments using yeast transketolase was 100 µM and therefore only saturating for TK high . As such, the two-step activation of transketolase was observed only in the formation of TK high , and may well be different for TK low . Additional experiments at higher [TPP] would be required to elucidate the mechanism of cofactor-binding and activation of TK low .

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Section 7: Supplementation of fermentation with thiamine Figure S6: Experimental data of 0.05 mg/mL wild-type transketolase binding to TPP at 9 mM Mg 2+ , purified from a fermentation supplemented with 0.5 mM thiamine. Experimental data-points were fitted to the double-Hill function (black). The %TK high was 34.5 + 15.8%, comparable to that of transketolase purified from un-supplemented fermentations.