Computer modeling of transketolase-like protein, TKTL1, a marker of certain tumor tissues.

A computer model of the spatial structure of transketolase-like protein (TKTL1), a marker of certain tumor tissues, has been constructed using the known spatial structure of transketolase found in normal human tissues. The structure of the two proteins at all levels of their organization has also been compared. On the basis of the revealed differences in structures of these proteins, we assume it is unlikely that TKTL1 can be a thiamine diphosphate-dependent protein capable of catalyzing the transketolase reaction.

Interaction of transketolase from human tissues with substrates.

The Michaelis constant values for substrates of transketolase from human tissues were determined over a wide range of substrate concentrations. It is shown that K(m) values determined by other authors are significantly overestimated and explained why this is so.

Inhibition of transketolase by hexacyanoferrate(III).

The effect of hexacyanoferrate(III) on the catalytic activity of transketolase has been studied. This oxidant inactivates only one of two active sites of the enzyme, the one with a higher affinity to the coenzyme (thiamine diphosphate). The second active site does not lose its catalytic activity. These observations indicate that the active sites of holotransketolase, being indiscernible by data of X-ray analysis, exhibit functional nonequivalence.

Isolation and properties of human transketolase.

Recombinant human (His)(6)-transketolase (hTK) was obtained in preparative amounts by heterologous expression of the gene encoding human transketolase in Escherichia coli cells. The enzyme, isolated in the form of a holoenzyme, was homogeneous by SDS-PAGE; a method for obtaining the apoenzyme was also developed. The amount of active transketolase in the isolated protein preparation was correlated with the content of thiamine diphosphate (ThDP) determined in the same preparation. Induced optical activity, facilitating studies of ThDP binding by the apoenzyme and measurement of the transketolase reaction at each stage, was detected by circular dichroism spectroscopy. A single-substrate reaction was characterized, catalyzed by hTK in the presence of the donor substrate and in the absence of the acceptor substrate. The values of the Michaelis constant were determined for ThDP and a pair of physiological substrates of the enzyme (xylulose 5-phosphate and ribose 5-phosphate).

Halogenated pyruvate derivatives as substrates of transketolase from Saccharomyces cerevisiae.

Pyruvate derivatives halogenated at C3 were shown to be donor substrates in the transketolase reaction. No drastic differences between the derivatives were observed in the value of the catalytic constant, whereas the Michaelis constant increased in the following order: Br-pyruvate < Cl-pyruvate < Cl2-pyruvate < F-pyruvate < Br2-pyruvate. The presence of the halogenated pyruvate derivatives increased the affinity of apotransketolase for the coenzyme; of note, the extent of this effect was equal with both of the active centers of the enzyme. In contrast, the presence of any other substrate known to date, including hydroxypyruvate (i.e. pyruvate hydroxylated at C3), induced nonequivalence of the active centers in that they differed in the extent to which the affinity for the coenzyme increased. Consequently, the beta-hydroxyl of dihydroxyethylthiamine diphosphate (an intermediate of the transketolase reaction) played an important role in the phenomenon of nonequivalence of the active centers associated with the coenzyme binding. The fundamental possibility was demonstrated of using halogenated pyruvate derivatives as donors of the halogen-hydroxyethyl group in organic synthesis of halogenated carbohydrates involving transketolase.

Cooperative binding of substrates to transketolase from Saccharomyces cerevisiae.

Catalytic activity of two active sites of transketolase and their affinity towards the substrates (xylulose-5-phosphate and ribose-5-phosphate) has been studied in the presence of Ca2+ and Mg2+. In the presence of Ca2+, the active sites exhibit negative cooperativity in binding both xylulose-5-phosphate (donor substrate) and ribose-5-phosphate (acceptor substrate) and positive cooperativity in the catalytic transformation of the substrates. In the presence of Mg2+, nonequivalence of the active sites is not observed.

New function of the amino group of thiamine diphosphate in thiamine catalysis.

In this work, we investigated the rate of formation of the central intermediate of the transketolase reaction with thiamine diphosphate (ThDP) or 4'-methylamino-ThDP as cofactors and its stability using stopped-flow spectroscopy and circular dichroism (CD) spectroscopy. The intermediates of the transketolase reaction were analyzed by NMR spectroscopy. The kinetic stability of the intermediate was shown to be dependent on the state of the amino group of the coenzyme. The rates of the intermediate formation were the same in the case of the native and methylated ThDP, but the rates of the protonation or oxidation of the complex in the ferricyanide reaction were significantly higher in the complex with methylated ThDP. A new negative band was detected in the CD spectrum of the complex transketolase--4'-methylamino-ThDP corresponding to the protonated dihydroxyethyl-4'-methylamino-ThDP released from the active sites of the enzyme. These data suggest that transketolase in the complex with the NH2-methylated ThDP exhibits dihydroxyethyl-4'-methylamino-ThDP-synthase activity. Thus, the 4'-amino group of the coenzyme provides kinetic stability of the central intermediate of the transketolase reaction, dihydroxyethyl-ThDP.

Which stage of the process of apotransketolase interaction with thiamine diphosphate is affected by the regulatory activity of the donor substrate?

The interaction of thiamine diphosphate (ThDP) with transketolase (TK) involves at least two stages: [formula: see text] During the first stage, an inactive intermediate complex (TK...ThDP) is formed, which is then transformed into a catalytically active holoenzyme (TK* - ThDP). The second stage is related to conformational changes of the protein. In the preceding publication (Esakova, O. A., Meshalkina, L. E., Golbik, R., Hübner, G., and Kochetov, G. A. Eur. J. Biochem. 2004, 271, 4189 - 4194) we reported that the affinity of ThDP for TK considerably increases in the presence of the donor substrate, which may be a mechanism whereby the activity of the enzyme is regulated under the conditions of the coenzyme deficiency. Here, we demonstrate that the substrate affects the stage of the reverse conformational transition, characterized by the constant k(-1): in the presence of the substrate, its value is decreased several fold, whereas K(d) and k(+1) remain unchanged.

Influence of donor substrate on kinetic parameters of thiamine diphosphate binding to transketolase.

The two-step mechanism of interaction of thiamine diphosphate (ThDP) with transketolase (TK) has been studied: TK + ThDP <--> TK...ThDP <--> TK*-ThDP. The scheme involves the formation of inactive intermediate complex TK...ThDP followed by its transformation into catalytically active holoenzyme, TK*-ThDP. The dissociation and kinetic constants for individual stages of this process have been determined. The values of forward and backward rate constants change in the presence of the donor substrate hydroxypyruvate. This finally leads to an increase in the overall affinity of the coenzyme to TK.

Two methods for determination of transketolase activity.

Two new optical methods for transketolase activity assay using only one substrate, xylulose 5-phosphate or glycol aldehyde, have been developed. For transketolase activity assay in the first method, it is necessary to add auxiliary enzyme, glyceraldehyde phosphate dehydrogenase. It is not needed in the second method. The range of transketolase concentration in the activity assay is 0.036-0.144 U/ml for the first method and 1.8-6.8 U/ml for the second one.

Binding of the coenzyme and formation of the transketolase active center.

Transketolase (TK) is a homodimer, the simplest representative of thiamine diphosphate (ThDP)-dependent enzymes. It was first ThDP-dependent enzymes the crystal structure of which has been solved and revealed the general fold for this class of enzymes and the interactions of the non-covalently bound coenzyme ThDP with the protein component. Transketolase is a convenient model to study the structure(s) of the active center and the mechanism of action of ThDP-dependent enzymes. This review summarizes the results of studies on the kinetics of the interaction of ThDP with TK from Saccharomyces cerevisiae as well as the generation of the catalytically active form of the coenzyme within the holoenzyme and formation of the enzyme's active center.

Effect of transketolase substrates on holoenzyme reconstitution and stability.

The influence of transketolase substrates on the interaction of apotransketolase with its coenzyme thiamine diphosphate (TDP) and on the stability of the reconstituted holoenzyme was studied. Donor substrates increased the affinity of the coenzyme for transketolase, whereas acceptor substrate did not. In the presence of magnesium ions, the active centers of transketolase initially identical in TDP binding lose their equivalence in the presence of donor substrates. The stability of transketolase depended on the cation type used during its reconstitution--the holoenzyme reconstituted in the presence of calcium ions was more stable than the holoenzyme produced in the presence of magnesium ions. In the presence of donor substrate, the holoenzyme stability increased without depending on the cation used during the reconstitution. Donor substrate did not influence the interaction of apotransketolase with the inactive analog of the coenzyme N3'-pyridyl thiamine diphosphate and did not stabilize the transketolase complex with this analog. The findings suggest that the effect of the substrate on the interaction of the coenzyme with apotransketolase and on stability of the reconstituted holoenzyme is caused by generation of 2-(alpha,beta-dihydroxyethyl)thiamine diphosphate (an intermediate product of the transketolase reaction), which has higher affinity for apotransketolase than TDP.

Optical characteristics of thiamine in model systems and in holoenzyme.

The optical properties of thiamine diphosphate-dependent enzymes change significantly on their interaction with cofactors (thiamine, bivalent metal ions) and substrates. These changes are connected with structural alterations of the active site and the mechanism of its functioning, and in some cases they reflect changes in the optical properties of the coenzyme itself within the protein. The use of optical characteristics, especially together with model systems, appeared to be a rather promising approach for investigation of the active site of thiamine diphosphate-dependent enzymes and the mechanism of its functioning. So, it seemed to be useful to summarize the literature data concerning the optical characteristics of thiamine (thiamine diphosphate) in model systems and the efficiency of their application for study of thiamine diphosphate-dependent enzymes.

Induced absorption band of holotransketolase and its interpretation.

It has long been known that formation of a catalytically active holotransketolase from the apoenzyme and thiamine diphosphate (ThDP) is accompanied by appearance, in both the absorption and CD spectra, of a new band. Binding and subsequent conversion of transketolase substrates bring about changes in the intensity of this band. The observation of these changes allows the investigator to monitor the coenzyme-to-apoenzyme binding and the conversion of the substrates during the transketolase reaction and thus to kinetically characterize its individual steps. As regards the new absorption band induced by ThDP binding, its nature, until recently, remained unknown. The reason for its appearance was considered to be either the formation of a charge transfer complex between ThDP and tryptophan (phenylalanine) residue or stacking interaction between the residues of aromatic amino acids. They are thought to be brought together as a result of conformational changes of the apoenzyme during its interaction with the coenzyme. However none of these hypotheses had been substantiated experimentally. According to our hypothesis, the induced absorption band is that of the imino form of ThDP resulting from three contributing features of the ThDP binding site of transketolase: the relative hydrophobicity of this site, hydrogen bonding of the N1;-atom of the ThDP aminopyrimidine ring to Glu418, and base stacking interactions between the aminopyrimidine ring of ThDP and Phe445.

Functional flexibility of the transketolase molecule.

Transketolase is the simplest representative of the thiamine diphosphate-dependent enzymes. It was the first of these enzymes for which X-ray analysis was performed. Based on the data of X-ray studies and using the mutagenesis technique, the nature of functional groups of the enzyme involved in the interaction with substrates and cofactors and in the coenzyme activation was defined. Thus, considerable achievements have been made in studying the structure of transketolase. However, there is relatively little information on the conformational flexibility of the enzyme molecule while it is functioning, i.e., during its interaction with cofactors and substrates and in the course of intermediate product formation. This review summarizes mainly the results obtained in the author's group, as well as those rare data on this subject that could be found in literature.

Cleaving of ketosubstrates by transketolase and the nature of the products formed.

The interaction of transketolase ketosubstrates with the holoenzyme has been studied. On addition of ketosubstrates cleaving both irreversibly (hydroxypyruvate) and reversibly (xylulose 5-phosphate), identical changes in the CD spectrum at 300-360 nm are observed. The changes in this spectral region, as previously shown, are due to the formation of the catalytically active holoenzyme from the apoenzyme and the coenzyme, and the cleavage of ketosubstrates by transketolase. The identity of the changes in transketolase CD spectrum caused by the addition of reversibly or irreversibly cleaving substrates indicates that in the both cases the changes are due to the formation of an intermediate product of the transketolase reaction--a glycolaldehyde residue covalently bound to the coenzyme within the holoenzyme molecule. Usually, in the course of the transferase reaction, the glycolaldehyde residue is transferred to an aldose (acceptor substrate), resulting in the recycling of the holoenzyme free of the glycolaldehyde residue. The removal of the glycolaldehyde residue from the holoenzyme appears to proceed even in the absence of an aldose. However, the glycolaldehyde cannot be found the free state because it condenses with another glycolaldehyde residue formed in the course of the cleavage of another ketosubstrate molecule yielding erythrulose.

One-substrate transketolase-catalyzed reaction.

Apart from catalyzing the common two-substrate reaction with ketose as donor substrate and aldose as acceptor substrate, transketolase is also able to catalyze a one-substrate reaction utilizing only ketose (xylulose 5-phosphate) as substrate. The products of this one-substrate reaction were glyceraldehyde 3-phosphate and erythrulose. No free glycolaldehyde (a product of xylulose 5-phosphate splitting in the transketolase reaction) was revealed.

Acceptor substrate inhibits transketolase competitively with respect to donor substrate.

Two substrates of the transketolase reaction are known to bind with the enzyme according to a ping-pong mechanism [1]. It is shown in this work that high concentrations of ribose-5-phosphate (acceptor substrate) compete with xylulose-5-phosphate (donor substrate), suppressing the transketolase activity (Ki = 3.8 mM). However, interacting with the donor-substrate binding site on the protein molecule, the acceptor substrate, unlike the donor substrate, does not cause any change in the active site of the enzyme. The data are interesting in terms of studying the regulatory mechanism of the transketolase activity and the structure of the enzyme-substrate complex.

Influence of transketolase substrates on its conformation.

Dynamics stimulation of the holotransketolase molecule revealed that the enzyme's conformation in crystal was different from that in solution. It was shown also that dissolved holotransketolase can bind aldose (the acceptor substrate) even in the absence of ketose (the donor substrate). The holotransketolase conformation did not change upon aldose binding unlike in the case of ketose binding/cleavage. Therefore the conformation of a catalytic complex of holotransketolase with an intermediate-i.e., a glycolaldehyde residue formed upon binding and subsequent cleavage of ketose-differed, at least in solution, from the conformation of both the free and aldose-complexed holotransketolase. Some structural peculiarities of the holotransketolase with the intermediate were established by means of molecular dynamics stimulation.

Inhibition of transketolase by p-hydroxyphenylpyruvate.

The effect of p-hydroxyphenylpyruvate, a natural analogue of transketolase substrate, on the catalytic activity of the enzyme was investigated. p-Hydroxyphenylpyruvate proved to be a reversible and competitive inhibitor of transketolase with respect to substrate; it was also able to displace thiamine diphosphate from holotransketolase. The data suggest that p-hydroxyphenylpyruvate participates in the regulation of tyrosine biosynthesis by influencing the catalytic activity of transketolase.