Marina V. Kovina

Kinetic mechanism of active site non-equivalence in transketolase.

The two‐step mechanism of coenzyme (TDP) binding to apotransketolase has been examined by kinetic modeling, and the rate and equilibrium constants for each binding step for two active sites have been determined. The dissociation constants for the primary fast binding step and the forward rate constants for the secondary slow binding step have been shown to be similar for two active sites. The backward rate constants for the secondary binding step are different for two active sites, providing the kinetic mechanism of their non‐equivalence in TDP binding.

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.

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.

Localization of reactive tyrosine residues of baker's yeast transketolase

Bakers yeast transketolase inactivated by tetranitromethane was digested with Staphylococcus aureus V8 protease. Four peptides absorbing at 360 nm were isolated by reverse‐phase HPLC and sequenced. The modified tyrosines were identified as Tyr‐184, Tyr‐210 and Tyr‐370.

The molecular origin of the thiamin diphosphate‐induced spectral bands of ThDP‐dependent enzymes

New and previously published data on a variety of ThDP‐dependent enzymes such as bakers 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. Proteins 2004.

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].

New in the mechanism of one-substrate transketolase reaction

Abstract 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.