Elsevier

Life Sciences

Volume 78, Issue 1, 19 November 2005, Pages 8-13
Life Sciences

Effects of transketolase cofactors on its conformation and stability

https://doi.org/10.1016/j.lfs.2004.12.055Get rights and content

Abstract

In studying transketolase (TK) from Saccharomyces cerevisiae, the majority of researchers use as cofactors Mg2+ and thiamine diphosphate (ThDP) (by analogy with other ThDP-dependent enzymes), whereas the active site of native holoTK is known to contain only Ca2+. Experiments in which Mg2+ was substituted for Ca2+ 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 Ca2+ or Mg2+, respectively, adopt different conformations [Selivanov, V.A., Kovina, M.V., Kochevova, N.V., Meshalkina, L.E., Kochetov, G.A., 2004. 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 Ca2+ or Mg2+, respectively, turned out to be different. The two forms of the holoenzyme, obtained by reconstitution from apoTK and ThDP in the presence of Ca2+ or Mg2+, respectively, also differed in stability: the holoenzyme was more stable in the presence of Ca2+ than Mg2+.

Introduction

Transketolase (TK; EC 2.2.1.1), a thiamine diphosphate (ThDP)-dependent enzyme, catalyzes one of the key reactions of the pentose phosphate pathway of carbohydrate transformation, i.e., the cleavage of the carbon–carbon bond adjacent to the carbonyl group of ketoses (donor substrates), with subsequent transfer of the two-carbon unit to aldosugars (acceptor substrates) (Kochetov, 1982). The enzyme is a homodimer with two active sites located at the interface between the contacting surfaces of the monomers (Lindqvist et al., 1992, Nikkola et al., 1994). All ThDP-dependent enzymes, including TK, require bivalent cations for their action. Magnesium ions are generally used as such cofactors in all investigations with ThDP-dependent enzymes. Structures of TK from maize (Gerhardt et al., 2003) and Leishmania mexicana (Veitch et al., 2004) in the presence of Mg2+ and ThDP were solved recently. Native holoTK from Saccharomyces cerevisiae was shown to contain Ca2+ (2 g atom per mol protein) (Kochetov and Philippov, 1970). ThDP is cleaved in the course of isolation and purification of the enzyme; addition of the coenzyme and bivalent cations is prerequisite to restoring the activity. Data of crystal structure analysis demonstrate that Ca2+ acts as a mediator between the protein and the anchoring portion of ThDP, the diphosphate residue (Lindqvist et al., 1992, Nikkola et al., 1994). In the presence of Ca2+, the structures of apo- and holoTK differ in the position of two loops (each loop is located in one of the two protein subunits of the enzyme) corresponding to residues 187–198 and 383–394: the loops are relatively flexible in the apoenzyme and structured in the holoenzyme (Lindqvist et al., 1992, Nikkola et al., 1994, Sundström et al., 1992). Certain properties of the holoenzyme reconstituted from apoTK, ThDP, and bivalent cations (Ca2+ or Mg2+) depend on the cation used in the course of reconstitution.

For example, in the presence of external Ca2+, ThDP binding is characterized by negative cooperativity (Egan and Sable, 1981, Kovina et al., 1997). The values of apparent constants of ThDP dissociation, measured for the two active sites, differ by approximately one order of magnitude. Of note, this difference between the sites is revealed only at the stage of the reverse conformational transition, the rate constant of which (k 1 in Fig. 1) in one of the active sites increases by approximately one order of magnitude (Kovina et al., 1997). In other words, the reciprocal effects of the sites become evident only after the two stages of ThDP binding have been completed in both active sites. Thus, structurally identical active sites of TK (Lindqvist et al., 1992, Nikkola et al., 1994) become non-equivalent in the process of ThDP binding (Kovina et al., 1997).

In the presence of external Mg2+, the affinity of the active sites of TK for the coenzyme is considerably lower than in the presence of Ca2+ (Egan and Sable, 1981, Heinrich et al., 1972a, Kochetov et al., 1975, Selivanov et al., 2003), and the cooperative effect is either weak (Selivanov et al., 2003) or absent (Heinrich et al., 1972a). The value of the rate constant of the reverse conformational transition (k 1 in Fig. 1) also depends on the type of the bivalent cation. In the presence of Mg2+, its value is approximately 6 times higher than in the presence of Ca2+, which accounts for the lower affinity of ThDP for apoTK with Mg2+, as compared to Ca2+ (Kovina et al., 1997, Selivanov et al., 2003).

Effects of Mg2+ and Ca2+ on ThDP interaction with wild-type (WT) apoTK or its H103A mutant provide yet another example of the dependence of TK properties on bivalent cation type (Selivanov et al., 2004). Substitution of alanine for histidine at position 103 fails to affect the coenzyme binding to apoTK in the presence of Ca2+. This observation is in agreement with data of crystal X-ray structure analysis, demonstrating lack of differences between WT holoTK and H103A mutant holoTK (both enzymes were reconstituted in the presence of Ca2+). In the presence of Mg2+, however, all kinetic characteristics of the process of holoTK reconstitution were changed, which produced a considerable decrease in the affinity of ThDP for the H103A mutant apoprotein (as compared to WT apoTK). These findings suggest that the structures of active sites of TK, formed in the presence of Mg2+ or Ca2+, are not identical.

Thus, according to the data obtained earlier, a variety of kinetic properties of TK essentially depend on the type of bivalent cation (Ca2+ or Mg2+) used as cofactor in the course of holoTK reconstitution (Egan and Sable, 1981, Selivanov et al., 2003, Selivanov et al., 2004). One of the reasons of observable distinctions is, apparently, various protein conformation, caused by the type of used cation. This work is a part of systematical research on the specificity of the influence of bivalent cations on the structure and kinetic properties of TK. We used a variety of methods to study the effects of bivalent cations on the conformational stability of TK (in the presence and absence of its coenzyme, ThDP).

Section snippets

Materials

ThDP, and glycyl–glycine were purchased from Serva; CaCl2 and MgCl2, from Fluka. The quality of other commercially available chemicals was no lower than reagent grade.

Transketolase purification

Recombinant baker's yeast TK with a specific activity of 22 U/mg was isolated by a method described previously (Wikner et al., 1994). The enzyme was obtained as apoTK, which was homogeneous by SDS-PAGE. TK concentration was determined spectrophotometrically, using A1cm1% of 14.5 at 280 nm (Heinrich et al., 1972b).

Measurement of ThDP concentration

ThDP

Results and discussion

All measurements were done in the presence of saturating concentration of bivalent cations − 2.5 mM Ca2+ or 2.5 mM Mg2+.

Conclusion

The conformations adopted by apoTK in the presence of Mg2+ or Ca2+, differ. Judged from the far-UV CD spectra (Fig. 2) of the enzyme, this conclusion is valid at least for the secondary structure. ThDP binding by the apoprotein (in the presence of either Mg2+ or Ca2+) fails to affect the secondary structure of TK, as indicated by the lack of changes in the CD spectrum. On the other hand, ThDP exerts a considerable stabilizing effect on apoTK, in agreement with data of perturbational

Acknowledgment

This work was supported by the Russian Foundation for Basic Research (Project No. 03-04-49025).

References (24)

  • V.A. Selivanov et al.

    Kinetic study of the H103A mutant yeast transketolase

    FEBS Letters

    (2004)
  • M. Sundström et al.

    Three-dimensional structure of apotransketolase

    FEBS Letters

    (1992)
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