A new method of determination of transketolase activity by asymmetric synthesis reaction

Kochetov, G. A.; Усманов, Р А; Mevkh, Alevtina T.

doi:10.1016/0003-2697(78)90422-0

Cited by 18 publications

(14 citation statements)

References 7 publications

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“…The results are given in Table III. Several authors have contributed to the determination of the substrate specificity of TK enzymes from different sources (E. coli, S. cerevisiae, G. stearothermophilus) using different assay methods towards various phosphorylated and unphosphorylated aldoses or aldehydes. 4,[14][15][16][17][18][19][20] The activity profiles of TKs are very similar, owing to the strong homology of active sites. Our system yields the same results as those obtained with TK gst using other methods.…”

Section: Determination Of Tk Acceptor Specificitymentioning

confidence: 99%

“…Measurement of chiral product formation by an optical method 16,17 is highly dependent on the substrate structure, and so not of generic utility. Determination of HPA depletion by near-UV spectroscopic monitoring 18 or HPLC (High Performance Liquid Chromatography) analysis [14][15][16][17][18][19][20] is hampered by low sensitivity or low throughput. Colorimetric determination of ketose formation with tetrazolium red-based oxidation is restricted to non-hydroxylated aldehyde acceptors such as propanal.…”

Section: Introductionmentioning

confidence: 99%

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Droplet millifluidics for kinetic study of transketolase

et al. 2016

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We present a continuous-flow reactor at the millifluidic scale coupled with an online, non-intrusive spectroscopic monitoring method for determining the kinetic parameters of an enzyme, transketolase (TK) used in biocatalysis for the synthesis of polyols by carboligation. The millifluidic system used is based on droplet flow, a well-established method for kinetic chemical data acquisition. The TK assay is based on the direct quantitative measurement of bicarbonate ions released during the transketolase-catalysed reaction in the presence of hydroxypyruvic acid as the donor, thanks to an irreversible reaction: bicarbonate ions react with phosphoenolpyruvate (PEP) in the presence of PEP carboxylase as the first auxiliary enzyme. The oxaloacetate formed is reduced to malate by NADH in the reaction catalysed by malate dehydrogenase as the second auxiliary enzyme. The extent of oxidation of NADH was measured by spectrophotometry at 340 nm. This system gives a direct, quantitative, generic method to evaluate the TK activity versus different substrates. We demonstrate the accuracy of this strategy to determine the enzymatic kinetic parameters and to study the substrate specificity of a thermostable TK from thermophilic microorganism Geobacillus stearothermophilus, offering promising prospects in biocatalysis. Millifluidic systems are useful in this regard as they can be used to rapidly evaluate the TK activity towards various substrates, and also different sets of conditions, identifying the optimal operating environment while minimizing resource consumption and ensuring high control over the operating conditions. Published by AIP Publishing. [http://dx

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Section: Determination Of Tk Acceptor Specificitymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Droplet millifluidics for kinetic study of transketolase

et al. 2016

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“…Spectrophotometric assays were then performed for most of the isoenzymes, following a similar strategy to . Enzymes were assayed spectrophotometrically through detection of NADPH or NADH, by using coupling reactions where needed, with the exception of ribulose-5-phosphate-3-epimerase (RPE1) and ribose-5-phosphate ketol isomerase (RKI1) which where assayed using circular dichroism (CD, [Kochetov et al, 1978]). Assays were coupled with enzyme(s) in which NAD(P) or NAD(P)H is a product or substrate so that its formation or consumption could be followed spectrophotometrically at 340 nm using an extinction coefficient of 6.62 /mM/cm, unless the reaction of a particular enzyme consumes or produces NADH or NADPH, in which case no coupling enzymes were needed.…”

Section: Kineticsmentioning

confidence: 99%

Enzyme characterisation and kinetic modelling of the pentose phosphate pathway in yeast

Messiha

Kent

Malys

et al. 2013

Preprint

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We present the quantification and kinetic characterisation of the enzymes of the pentose phosphate pathway in Saccharomyces cerevisiae. The data are combined into a mathematical model that describes the dynamics of this system and allows for predicting changes in metabolite concentrations and fluxes in response to perturbations. We use the model to study the response of yeast to a glucose pulse. We then combine the model with an existing glycolysis one to study the effect of oxidative stress on carbohydrate metabolism. The combination of these two models was made possible by the standardized enzyme kinetic experiments carried out in both studies. This work demonstrates the feasibility of constructing larger network models by merging smaller pathway models.

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“… c Assay (a) is the activity test according to Kochetov, detecting glyceraldehyde 3‐phosphate by using xylulose 5‐phosphate and ribose 5‐phosphate as substrates [14]. d Assay (b) is the activity test of erythrulose formation on simultaneous incubation of hydroxypyruvate (donor) and glycolaldehyde (acceptor) monitored by near‐UV CD, as described in the Experimental procedures [15]. e Assay (c) is the activity test according to Usmanov & Kochetov, using hydroxypyruvate as the donor substrate and ferricyanide as the artificial electron acceptor [18].…”

Section: Near‐uv CD Spectramentioning

confidence: 99%

“…In both cases only the covalent intermediate, DHEThDP, could be detected by 1 H NMR according to the method established by Tittmann et al [19]. Given the ability of glycolaldehyde to react with the enzyme-bound a-carbanion ⁄ enamine of DHEThDP to form erythrulose [15][16][17], in a double-jump experiment the artificial donor substrate, hydroxypyruvate, was first incubated with the native holoenzyme in an equimolar ratio to the active site concentration for 8 s to obtain the maximum amplitude of absorbance at 300 nm, mentioned above. In the second jump the addition of glycolaldehyde (acceptor) to a final concentration of 100 lm caused a decrease in absorbance in a single exponential first-order reaction with an observed rate constant of 0.48 s )1 (Fig.…”

Section: Transient Kinetics Of Donor Substrate Conversionmentioning

confidence: 99%

Effect of coenzyme modification on the structural and catalytic properties of wild‐type transketolase and of the variant E418A from Saccharomyces cerevisiae

et al. 2005

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Transketolase from baker's yeast is a thiamin diphosphate-dependent enzyme in sugar metabolism that reconstitutes with various analogues of the coenzyme. The methylated analogues (4¢-methylamino-thiamin diphosphate and N1¢-methylated thiamin diphosphate) of the native cofactor were used to investigate the function of the aminopyrimidine moiety of the coenzyme in transketolase catalysis. For the wild-type transketolase complex with the 4¢-methylamino analogue, no electron density was found for the methyl group in the X-ray structure, whereas in the complex with the N1¢-methylated coenzyme the entire aminopyrimidine ring was disordered. This indicates a high flexibility of the respective parts of the enzyme-bound thiamin diphosphate analogues. In the E418A variant of transketolase reconstituted with N1¢-methylated thiamin diphosphate, the electron density of the analogue was well defined and showed the typical V-conformation found in the wild-type holoenzyme [Lindqvist Y, Schneider G, Ermler U, Sundstrøm M (1992) EMBO J 11, 2373-2379]. The near-UV CD spectrum of the variant E418A reconstituted with N1¢-methylated thiamin diphosphate was identical to that of the wild-type holoenzyme, while the CD spectrum of the variant combined with the unmodified cofactor did not overlap with that of the native protein. The activation of the analogues was measured by the H ⁄ D-exchange at C2. Methylation at the N1¢ position of the cofactor activated the enzyme-bound cofactor analogue (as shown by a fast H ⁄ D-exchange rate constant). The absorbance changes in the course of substrate turnover of the different complexes investigated (transient kinetics) revealed the stability of the a-carbanion ⁄ enamine as the key intermediate in cofactor action to be dependent on the functionality of the 4-aminopyrimidine moiety of thiamin diphosphate.

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A new method of determination of transketolase activity by asymmetric synthesis reaction

Cited by 18 publications

References 7 publications

Droplet millifluidics for kinetic study of transketolase

Droplet millifluidics for kinetic study of transketolase

Enzyme characterisation and kinetic modelling of the pentose phosphate pathway in yeast

Effect of coenzyme modification on the structural and catalytic properties of wild‐type transketolase and of the variant E418A from Saccharomyces cerevisiae

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