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

Abstract

We recently characterised a low-activity form of E. coli transketolase, tK low , 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 high , in the presence of Mg 2+. We observed previously that partial oxidation was responsible for increased tK high activity, while lowactivity tK low 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 high :tK low ratio that matched those observed previously via tpp binding to each variant. the HpA dissociation constant of tK low was comparable to the substrate-inhibition dissociation constant, K i HPA , determined previously. We provide evidence that K i HPA is a convolution of binding to the low-activity tK low-tK low dimer, and the tK low subunit of the partially-active tK high-tK low mixed dimer, where HpA binding to the tK low subunit of the mixed dimer results in inhibition of the active tK high subunit. Heat-activation of transketolase was similarly investigated and found to convert the tK low subunit of the mixed dimer to have tK high-like properties, but without oxidation. Transketolase is a key enzyme of the pentose phosphate pathway (PPP), is ubiquitous in all organisms, and provides a unique link between glycolysis and the non-oxidative phase of the PPP. Transketolase is a thiamine pyrophosphate (TPP)-dependent enzyme that reversibly transfers a two-carbon ketol group from a donor substrate (usually a five-carbon ketose) to an acceptor aldehyde substrate (usually a five-carbon aldose) via a ping-pong reaction mechanism, forming a new asymmetric CC bond with high regio-and stereo-specificity. In biocatalysis, β-hydroxypyruvate (HPA) is often used as the donor substrate due to the irreversible, concomitant release of CO 2 as a by-product. Strong substrate inhibition has been observed above 25 mM HPA with an inhibition constant of around 42 mM 1,2. The cause of this substrate inhibition is currently unknown and is addressed in this study. The inactive, apo-form of transketolase is in a monomer-dimer equilibrium that is dependent on protein concentration. Upon cofactor binding, both the inactive apo-monomer and apo-dimer are converted into the catalytically active, dimeric holo-form of, until recently, seemingly structurally-identical subunits, with two active-sites per homodimer, located at the subunit interface 3,4. Each active site is comprised of one divalent cation, such as Mg 2+ , and one TPP molecule. In the S. cerevisiae transketolase apo-dimer, even after removal of free Ca 2+ , one Ca 2+ ion was bound extremely tightly to one active site and could only be removed using harsh treatment, while the second Ca 2+ ion dissociated easily. Subs...

Fine‐tuning the activity and stability of an evolved enzyme active‐site through noncanonical amino‐acids

“…Each mass spectrum (n = 3; average spectra in black) was fitted to the sum of multiple Gaussian functions (smaller individual peaks) corresponding to unmodified (TK low ), singleand double-oxidised forms (TK high or TK active ) of the misincorporated species 385Q (red) and 385F (magenta); and TK low , TK high and the overoxidised, inactive forms (TK inactive ) of the ncAA-incorporated species 385pCNF (blue). These same species have been observed previously in both wild-type TK [8] and variant S385Y/D469T/R520Q [40]. The ratio of the peak area of unmodified, singly-and doubly oxidised peaks of Y and F was determined from their mass spectra (Fig.…”

“…The major caveat to Analysis 2 is that it averages out effects that may have arisen from any population of mixed dimers between the monomeric subunits of different subspecies. It also ignores any potential influence of the variant upon the ratio of TK high :TK low [40], which could in turn affect the overall activity, as well as the inhibition of TK high by TK low in the resulting mixed dimer species.…”

“…However, the proportion of variant pAMF that existed as the TK inactive subspecies of 385Q, 385F and 385Y was negligible and hence excluded from all calculations and from each mass spectra since they were dwarfed by the larger TK low peaks. However, formation of TK high is a redox-regulated cellular process and the ratio of TK high :TK low is relatively constant but variant-specific [40]. We therefore assumed that a) the two TK high peaks of each TK misincorporated species were present and still contributed towards the overall peak area of the mass spectra; and b) the ratio of TK high :TK low was equivalent in naturally incorporated and misincorporated natural amino acid species.…”

“…The major caveat to Analysis 2 is that it averages out effects that may have arisen from any population of mixed dimers between the monomeric subunits of different subspecies. It also ignores any potential influence of the variant upon the ratio of TK high :TK low [40], which could in turn affect the overall activity, as well as the inhibition of TK high by TK low in the resulting mixed dimer species. Analysis 3: Deconvolution of (nc)AA-TK active subspecies parameters Analysis 3 provided the most detailed information about the 'true' parameters of only the active subspecies within each (nc)AA-incorporated TK variant, (nc)AA-TK active (visualised in Fig.…”

“…However, understanding and potentially controlling the underlying heterogeneity and its impact on performance is still important. For example, we now know that TK exists as multiple homodimeric species [8,40], and yet all previous work evaluating the performance of TK variants in biocatalytic processes is no less useful or valid. Instead, the performance measured characterises the 'apparent' activity and stability of that variant as a biocatalyst, including all unmodified and modified species within the sample, obtained using a particular production process.…”