L. E. Meshalkina

Function of the aminopyrimidine part in thiamine pyrophosphate enzymes

Abstract To answer the question on the mechanistic significance of the pyrimidine moiety of thiamine pyrophosphate (TPP), the two pyridine analogs of TPP ( N 1 -pyridyl-TPP and N 3 -pyridyl-TPP), as well as 4′-deamino-TPP, have been resynthesized and incubated with the apoenzymes of pyruvate decarboxylase, pyruvate dehydrogenase complex, and transketolase. By comparison of activity and binding properties of the three TPP analogs it is shown that only N 1 -pyridyl-TPP causes catalytic activity (between 65 and 100%) with all the enzymes tested. N 3 -Pyridyl-TPP as well as 4′-deamino-TPP proved inactive generally. The binding experiments demonstrate that both analogs with the N 1 -atom preserved in the structure ( N 1 -pyridyl-TPP and 4′-deamino-TPP) offer practically the same affinity as TPP to the three apoenzymes tested. A mechanism is proposed that explains the essential function of the amino group and the pyrimidine- N i in TPP catalysis.

Studies of thiamin diphosphate binding to the yeast apotransketolase

Abstract Previously it was shown that the binding of thiamin diphosphate proceeds through two steps: fast primary binding and the subsequent slow conformational transition of the apoprotein. In the presence of Ca 2+ , the coenzyme binding occurs with negative cooperativity—owing to the increased rate of the reverse conformational transfer in one of the active centers after completion of ThDP binding at both active centers. There are three viewpoints on the enzyme behavior upon replacement of Ca 2+ with Mg 2+ : (a) negative cooperativity between the two centers is retained; (b) turns positive; (c) totally disappears. In this work, a comparative investigation of the interaction between ThDP and apotransketolase was undertaken and the negative cooperativity between the two centers in the presence of Mg 2+ , just as in the presence of Ca 2+ was demonstrated—albeit with the former cation it was somewhat less pronounced. The negative cooperativity with Mg 2+ , just as with Ca 2+ , was caused by an increase in the rate of reverse conformational transfer after the ThDP binding completion in both active centers.

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, α,β‐dihydroxyethyl‐thiamin diphosphate, by forming a hydrogen bond with the oxygen of its β‐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 Ca2+ (a cation found in the native holoenzyme) but changes all the kinetic parameters of the ThDP–apoTK interaction in the presence of Mg2+ (a cation commonly used in ThDP‐dependent enzymes studies). It was concluded that the structures of TK active centers formed in the presence of Mg2+ and Ca2+ 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.

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.

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

Transketolase from bakers 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 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 α‐carbanion/enamine as the key intermediate in cofactor action to be dependent on the functionality of the 4‐aminopyrimidine moiety of thiamin diphosphate.

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.

Inhibition of transketolase by analogues of the coenzyme

Abstract The effect of thiamine, thiamine monophosphate, pyrophosphate and thiazole pyrophosphate on the enzymatic activity of transketolase has been studied. All these compounds have been proved to inhibit the enzyme by competing with the coenzyme (thiamine pyrophosphate) for apotransketolase. The experimental data obtained indicate that the interaction of thiamine pyrophosphate with apoenzyme occurs at least in three points and at the expense of the thiamine moiety of the coenzyme molecule and its phosphate residues.

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.

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 β-hydroxyl of dihydroxyethylthiamine diphosphate (an intermediate of the transketolase reaction) played an important role in the phenomenon of non-equivalence 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.