The mechanism of a one-substrate transketolase reaction.

Transketolase catalyzes the transfer of a glycolaldehyde residue from ketose (the donor substrate) to aldose (the acceptor substrate). In the absence of aldose, transketolase catalyzes a one-substrate reaction that involves only ketose. The mechanism of this reaction is unknown. Here, we show that hydroxypyruvate serves as a substrate for the one-substrate reaction and, as well as with the xylulose-5-phosphate, the reaction product is erythrulose rather than glycolaldehyde. The amount of erythrulose released into the medium is equimolar to a double amount of the transformed substrate. This could only be the case if the glycol aldehyde formed by conversion of the first ketose molecule (the product of the first half reaction) remains bound to the enzyme, waiting for condensation with the second molecule of glycol aldehyde. Using mass spectrometry of catalytic intermediates and their subsequent fragmentation, we show here that interaction of the holotransketolase with hydroxypyruvate results in the equiprobable binding of the active glycolaldehyde to the thiazole ring of thiamine diphosphate and to the amino group of its aminopyrimidine ring. We also show that these two loci can accommodate simultaneously two glycolaldehyde molecules. It explains well their condensation without release into the medium, which we have shown earlier.

Substrate inhibition of transketolase.

We studied the influence of the acceptor substrate of transketolase on the activity of the enzyme in the presence of reductants. Ribose-5-phosphate in the presence of cyanoborohydride decreased the transketolase catalytic activity. The inhibition is caused by the loss of catalytic function of the coenzyme-thiamine diphosphate. Similar inhibitory effect was observed in the presence of NADPH. This could indicate its possible regulatory role not only towards transketolase, but also towards the pentose phosphate pathway of carbohydrate metabolism overall, taking into account the fact that it inhibits not only transketolase but also another enzyme of the pentose phosphate pathway--glucose 6-phosphate dehydrogenase [Eggleston L.V., Krebs H.A. Regulation of the pentose phosphate cycle, Biochem. J. 138 (1974) 425-435].

Structure and functioning mechanism of transketolase.

Studies of thiamine diphosphate-dependent enzymes appear to have commenced in 1937, with the isolation of the coenzyme of yeast pyruvate decarboxylase, which was demonstrated to be a diphosphoric ester of thiamine. For quite a long time, these studies were largely focused on enzymes decarboxylating α-keto acids, such as pyruvate decarboxylase and pyruvate dehydrogenase complexes. Transketolase, discovered independently by Racker and Horecker in 1953 (and named by Racker) [1], did not receive much attention until 1992, when crystal X-ray structure analysis of the enzyme from Saccharomyces cerevisiae was performed [2]. These data, together with the results of site-directed mutagenesis, made it possible to understand in detail the mechanism of thiamine diphosphate-dependent catalysis. Some progress was also made in studies of the functional properties of transketolase. The last review on transketolase, which was fairly complete, appeared in 1998 [3]. Therefore, the publication of this paper should not seem premature.

Is transketolase-like protein, TKTL1, transketolase?

Until recently it was assumed that the transketolase-like protein (TKTL1) detected in the tumor tissue, is catalytically active mutant form of human transketolase (hTKT). Human TKT shares 61% sequence identity with TKTL1. And the two proteins are 77% homologous at the amino acid level. The major difference is the absence of 38 amino acid residues in the N-terminal region of TKTL1. Site-specific mutagenesis was used for modifying hTKT gene; the resulting construct had a 114-bp deletion corresponding to a deletion of 38 amino acid residues in hTKT protein. Wild type hTKT and mutant variant (DhTKT) were expressed in Escherichia coli and isolated using Ni-agarose affinity chromatography. We have demonstrated here that DhTKT is devoid of transketolase activity and lacks bound thiamine diphosphate (ThDP). In view of these results, it is unlikely that TKTL1 may be a ThDP-dependent protein capable of catalyzing the transketolase reaction, as hypothesized previously.

Effects of free Ca²âº on kinetic characteristics of holotransketolase.

Catalytic activity has been demonstrated for holotransketolase in the absence of free bivalent cations in the medium. The two active centers of the enzyme are equivalent in both the catalytic activity and the affinity for the substrates. In the presence of free Ca²âº (added to the medium from an external source), this equivalence is lost: negative cooperativity is induced on binding of either xylulose 5-phosphate (donor substrate) or ribose 5-phosphate (acceptor substrate), whereupon the catalytic conversion of the bound substrates causes the interaction between the centers to become positively cooperative. Moreover, the enzyme total activity increase is observed.

Functional nonequivalence of transketolase active centers.

Transketolase (TK, EC 2.2.1.1), the key enzyme of the non-oxidative branch of pentose phosphate pathway of hydrocarbon transformation, plays an important role in a system of substrate rearrangement between pentose shunt and glycolysis, acting as a reversible link between the two metabolic pathways. In addition, it supplies precursors for biosyntheses of nucleotides, aromatic amino acids, and vitamins. In plants, the enzyme plays a central role in the Calvin cycle. TK catalyzes interconversion of sugar phosphates. Thiamine diphosphate (TDP) and bivalent cations serve as its cofactors. Being a typical TDP-dependent enzyme, TK is the least complex representative of this group of enzymes, and this accounts for its use as a model in studies of their structure and mechanism of action. TK is readily crystallized, this being the reason why the first crystal X-ray structure analysis of TDP-dependent enzymes was performed with a TK sample. Both the general structure of TK and the structures of its active centers have been studied in detail. In this article, we review experimental evidence of functional nonequivalence of the two active centers of TK, which are known to be identical by crystal X-ray structure analysis.

Effect of bivalent cations on the interaction of transketolase with its donor substrate.

The effect of the type of the cation cofactor of transketolase (i.e., Ca2+ or Mg2+) on its interaction with xylulose 5-phosphate (donor substrate) has been studied. In the presence of magnesium, the active centers of the enzyme were functionally equivalent with respect to xylulose 5-phosphate binding and exhibited identical affinities for the donor substrate. Substitution of Ca2+ for Mg2+ results in the loss of the equivalence. In particular, this becomes apparent on binding of xylulose 5-phosphates to one of the two active centers of the enzyme, which caused the second center to undergo a several fold decrease in the affinity for the donor substrate.

New evidence for cofactor's amino group function in thiamin catalysis by transketolase.

Transketolase from Saccharomyces cerevisiae exhibits a rarely reported activity with a methylated analogue of the native cofactor, 4'-methylamino-thiamin diphosphate. We demonstrated the kinetic stability of the dihydroxyethyl carbanion/enamine intermediate to be dependent on the functionality of the 4'-aminopyrimidine moiety of thiamin diphosphate [R. Golbik, L.E. Meshalkina, T. Sandalova, K. Tittmann, E. Fiedler, H. Neef, S. König, R. Kluger, G.A. Kochetov, G. Schneider, G. Hübner, Effect of coenzyme modification on the structural and catalytic properties of wild-type transketolase and of the variant E418A from Saccharomyces cerevisae, FEBS J. (2005) 272 1326-1342]. This paper extends these investigations of the function of the coenzyme's aminopyrimidine in transketolase catalysis exemplified for the 4'-monomethylamino-thiamin diphosphate analogue. Here, we report near UV circular dichroism data and NMR-based analysis of reaction intermediates that give evidence for a strong destabilisation of the carbanion/enamine of DHE-4'-monomethylamino-thiamin diphosphate on the enzyme. A new negative band in near UV circular dichroism arising during turnover is attributed to the conjugate acid of the carbanion/enamine intermediate, an assignment additionally corroborated by (1)H NMR-based intermediate analysis. As opposed to the kinetically stabilized carbanion/enamine intermediate in transketolase when reconstituted with the native cofactor, DHE-4'-monomethylamino-thiamin diphosphate is rapidly released from the active centers during turnover and accumulates in the medium on a preparative scale.

Nonequivalence of transketolase active centers with respect to acceptor substrate binding.

The interaction of transketolase with its acceptor substrate, ribose 5-phosphate, has been studied. The active centers of the enzyme were shown to be functionally nonequivalent with respect to ribose 5-phosphate binding. Under the conditions where only one out of the two active centers of transketolase is functional, their affinities for ribose 5-phosphate are identical. The phenomenon of nonequivalence becomes apparent when the substrate interacts with one of the two active centers. As a consequence of such interaction, the affinity of the second active center for ribose 5-phosphate decreases.

Effects of transketolase cofactors on its conformation and stability.

In studying transketolase (TK) from Saccharomyces cerevisiae, the majority of researchers use as cofactors Mg(2+) and thiamine diphosphate (ThDP) (by analogy with other ThDP-dependent enzymes), whereas the active site of native holoTK is known to contain only Ca(2+). Experiments in which Mg(2+) was substituted for Ca(2+) demonstrated that the kinetic properties of TK varied with the bivalent cation cofactor. This led to the assumption that TK species obtained by reconstitution from apoTK and ThDP in the presence of Ca(2+) or Mg(2+), respectively, adopt different conformations. Kinetic study of the H103A mutant yeast transketolase. FEBS Letters 567, 270-274]. Analysis of far-UV circular dichroism (CD) spectra and of data, obtained using methods of thermal denaturing, differential scanning calorimetry (DSC) and tryptophan fluorescence spectroscopy, corroborated this assumption. Indeed, the ratios of secondary structure elements in the molecule of apoTK, recorded in the presence of Ca(2+) or Mg(2+), respectively, turned out to be different. The two forms of the holoenzyme, obtained by reconstitution from apoTK and ThDP in the presence of Ca(2+) or Mg(2+), respectively, also differed in stability: the holoenzyme was more stable in the presence of Ca(2+) than Mg(2+).

Rapid simulation and analysis of isotopomer distributions using constraints based on enzyme mechanisms: an example from HT29 cancer cells.

MOTIVATION: Addition of labeled substrates and the measurement of the subsequent distribution of the labels in isotopomers in reaction networks provide a unique method for assessing metabolic fluxes in whole cells. However, owing to insufficiency of information, attempts to quantify the fluxes often yield multiple possible sets of solutions that are consistent with a given experimental pattern of isotopomers. In the study of the pentose phosphate pathways, the need to consider isotope exchange reactions of transketolase (TK) and transaldolase (TA) (which in past analyses have often been ignored) magnifies this problem; but accounting for the interrelation between the fluxes known from biochemical studies and kinetic modeling solves it. The mathematical relationships between kinetic and equilibrium constants restrict the domain of estimated fluxes to the ones compatible not only with a given set of experimental data, but also with other biochemical information. METHOD: We present software that integrates kinetic modeling with isotopomer distribution analysis. It solves the ordinary differential equations for total concentrations (accounting for the kinetic mechanisms) as well as for all isotopomers in glycolysis and the pentose phosphate pathway (PPP). In the PPP the fluxes created in the TK and TA reactions are expressed through unitary rate constants. The algorithms that account for all the kinetic and equilbrium constant constraints are integrated with the previously developed algorithms, which have been further optimized. The most time-consuming calculations were programmed directly in assembly language; this gave an order of magnitude decrease in the computation time, thus allowing analysis of more complex systems. The software was developed as C-code linked to a program written in Mathematica (Wolfram Research, Champaign, IL), and also as a C++ program independent from Mathematica. RESULTS: Implementing constraints imposed by kinetic and equilibrium constants in the isotopomer distribution analysis in the data from the cancer cells eliminated estimates of fluxes that were inconsistent with the kinetic mechanisms of TK and TA. Fluxes measured experimentally in cells can be used to estimate better the kinetics of TK and TA as they operate in situ. Thus, our approach of integrating various methods for in situ flux analysis opens up the possibility of designing new types of experiments to probe metabolic interrelationships, including the incorporation of additional biochemical information. AVAILABILITY: Software is available freely at: http://www.bq.ub.es/bioqint/selivanov.htm CONTACT: martacascante@ub.edu

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

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, Sundstrom M (1992) EMBO J11, 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 alpha-carbanion/enamine as the key intermediate in cofactor action to be dependent on the functionality of the 4-aminopyrimidine moiety of thiamin diphosphate.

Donor substrate regulation of transketolase.

The influence of substrates on the interaction of apotransketolase with thiamin diphosphate was investigated in the presence of magnesium ions. It was shown that the donor substrates, but not the acceptor substrates, enhance the affinity of the coenzyme either to only one active center of transketolase or to both active centers, but to different degrees in each, resulting in a negative cooperativity for coenzyme binding. In the absence of donor substrate, negative cooperativity is not observed. The donor substrate did not affect the interaction of the apoenzyme with the inactive coenzyme analogue, N3'-pyridyl-thiamin diphosphate. The influence of the donor substrate on the coenzyme-apotransketolase interaction was predicted as a result of formation of the transketolase reaction intermediate 2-(alpha,beta-dihydroxyethyl)-thiamin diphosphate, which exhibited a higher affinity to the enzyme than thiamin diphosphate. The enhancement of thiamin diphosphate's affinity to apotransketolase in the presence of donor substrate is probably one of the mechanisms underlying the substrate-affected transketolase regulation at low coenzyme concentrations.

Kinetic study of the H103A mutant yeast transketolase.

Data from site-directed mutagenesis and X-ray crystallography show that His103 of holotransketolase (holoTK) does not come into contact with thiamin diphosphate (ThDP) but stabilizes the transketolase (TK) reaction intermediate, alpha,beta-dihydroxyethyl-thiamin diphosphate, by forming a hydrogen bond with the oxygen of its beta-hydroxyethyl group [Eur. J. Biochem. 233 (1995) 750; Proc. Natl. Acad. Sci. USA 99 (2002) 591]. We studied the influence of His103 mutation on ThDP-binding and enzymatic activity. It was found that mutation does not affect the affinity of the coenzyme to apotransketolase (apoTK) in the presence of Ca(2+) (a cation found in the native holoenzyme) but changes all the kinetic parameters of the ThDP-apoTK interaction in the presence of Mg(2+) (a cation commonly used in ThDP-dependent enzymes studies). It was concluded that the structures of TK active centers formed in the presence of Mg(2+) and Ca(2+) are not identical. Mutation of His103 led to a significant acceleration of the one-substrate reaction but a slow down of the two-substrate reaction so that the rates of both types of catalysis became equal. Our results provide evidence for the intermediate-stabilizing function of His103.

The molecular origin of the thiamine diphosphate-induced spectral bands of ThDP-dependent enzymes.

New and previously published data on a variety of ThDP-dependent enzymes such as baker's yeast transketolase, yeast pyruvate decarboxylase and pyruvate dehydrogenase from pigeon breast muscle, bovine heart, bovine kidney, Neisseria meningitidis and E. coli show their spectral sensitivity to ThDP binding. Although ThDP-induced spectral changes are different for different enzymes, their universal origin is suggested as being caused by the intrinsic absorption of the pyrimidine ring of ThDP, bound in different tautomeric forms with different enzymes. Non-enzymatic models with pyrimidine-like compounds indicate that the specific protein environment of the aminopyrimidine ring of ThDP determines its tautomeric form and therefore the changeable features of the inducible effect. A polar environment causes the prevalence of the aminopyrimidine tautomeric form (short wavelength region is affected). For stabilization of the iminopyrimidine tautomeric form (both short- and long-wavelength regions are affected) two factors appear essential: (i) a nonpolar environment and (ii) a conservative carboxyl group of a specific glutamate residue interacting with the N1' atom of the aminopyrimidine ring. The two types of optical effect depend in a different way upon the pH, in full accordance with the hypothesis tested. From these studies it is concluded that the inducible optical rotation results from interaction of the aminopyrimidine ring with its asymmetric environment and is defined by the protonation state of N1' and the 4'-nitrogen.

A hitherto unknown transketolase-catalyzed reaction.

Yeast transketolase, in addition to catalyzing the transferase reaction through utilization of two substrates--the donor substrate (ketose) and the acceptor substrate (aldose)--is also able to catalyze a one-substrate reaction with only aldose (glycolaldehyde) as substrate. The interaction of glycolaldehyde with holotransketolase results in formation of the transketolase reaction intermediate, dihydroxyethyl-thiamin diphosphate. Then the glycolaldehyde residue is transferred from dihydroxyethyl-thiamin diphosphate to free glycolaldehyde. As a result, the one-substrate transketolase reaction product, erythrulose, is formed. The specific activity of transketolase was found to be 0.23 U/mg and the apparent Km for glycolaldehyde was estimated as 140 mM.

The origin of the absorption band induced through the interaction between apotransketolase and thiamin diphosphate.

It has long been known that formation of a catalytically active holotransketolase from the apoenzyme and coenzyme (thiamin diphosphate) is accompanied by the appearance of a new band, in both the absorption and CD spectra. Binding and subsequent conversion of the substrates bring about changes in this band's intensity. The observation of these changes allows the investigator to monitor the coenzyme-to-apoenzyme binding and the conversion of substrates during the transketolase reaction and thus to kinetically characterize its individual steps. The origin of the thiamin diphosphate induced absorption band has been postulated to be resulted from formation of a charge transfer complex or alternatively from an induced conformational transition of the enzyme. The latter brings aromatic amino acid residues into close proximity and generates the absorption. However, X-ray crystallographic and enzyme point mutation experiments cast doubts on both of these hypotheses. Here we show that the binding of thiamin diphosphate to the apotransketolase leads to the conversion of the 4'-amino tautomeric form of its aminopyrimidine ring into the N(1')H-imino tautomeric form. This imino form emerges as a result of the coenzyme's aminopyrymidine ring incorporation into the hydrophobic pocket of the transketolase active center and is stabilized through the interactions with Glu418 and Phe445 residues. The N(1')H-imino tautomeric form of thiamin diphosphate is thought to be the origin of the holotransketolase absorption band induced through the coenzyme binding.