Kochetov Ga

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.

Thiaminepyrophosphate induced changes in the optical activity of baker's yeast transketolase

Induced optical activity results from a type of asymmetry arising from the spatial orientation of some limited sites of the protein molecule and is revealed by the presence of chromophores*. Different types of substances act as chromophores in inducing OA * * on interaction with protein: the haem prosthetic group; FAD; NAD; pyridoxalphosphate and their derivatives; metallic ions, e.g. Fe3+, Mn3+, Cu2+, Cd2+; specific substrates, inhibitors and some dyes [ 1,2] . It is interesting to study optically active side-chain chromophores which are part of the protein molecule [3] . . The stereospecificity of OA-inducing chromophores and the fact that induced OA does not depend on OA of the peptide chromophore makes the phenomenon of induced OA a valuable eye witness of events occurring at the enzyme active centre. No data exist about the OA of enzymes with TPP as coenzymes. We have previously studied the OA of one of the thiamine enzymes, baker’s yeast transketolase, by means of ORD [4] . This paper reports the results of changes in OA of transketolase arising on TPP interaction with apoenzyme. Transketolase (EC 2.2. I. 1) was prepared from baker’s yeast as described [f] _ The specific activity of TK preparations was 10 U/mg. The enzyme produced a single symmetrical peak in the ultracentrifuge

The functional identity of the active centres of transketolase.

Direct determination of the number of catalytically active molecules of the coenzyme in holotransketolase (sedoheptulose-7-phosphate:D-glyceraldehyde-3-phosphate glycoaldehydetransferase, EC 2.2.1.1) has corroborated our previous data indicating that in the native enzyme there are two active centres. They have been provided to be functionally identical. It has been shown that the decrease in the specific activity of transketolase during its storage is due to inactivation of one of the active centres, having a lower affinity for the coenzyme. The second active centre retains thereby its full catalytic activity.

Charge transfer interactions in transketolase-thiamine pyrophosphate complex.

Abstract Absorption spectra of apo- and holotransketolase were measured and the results were compared with those of model systems. This comparison suggests that thiamine pyrophosphate, coenzyme of transketolase, interacts with tryptophanyl residue in the active centre of apoenzyme. The mechanism involved is supposed to be that of charge transfer complex formation.

The number of active sites in a molecule of transketolase

Abstract It has been demonstrated that the previously described changes in the optical properties of apotransketolase interacting with thyamine pyrophosphate are associated only with the catalytically active molecules of coenzyme being bound. Titration of apoenzyme by TPP has shown a molecule of transketolase to have two active centres.

The binding of thiamine pyrophosphate with transketolase in equilibrium conditions

Abstract The binding between thiamine pyrophosphate and transketolase, purified from bakers yeast, in equilibrium conditions has been studied. In the presence of Ca2+, the enzyme molecule has been shown to possess two binding sites for the coenzyme, whose dissociation constants are 3.2 × 10−8 and 2.5 × 10−7M; besides, there are site(s) where the binding of the coenzyme is less firm. In the presence of Mg2+, a positive cooperative interaction between the binding sites of thiamine pyrophosphate has been observed. Regardless of the cation used, the major part of the catalytic activity of the transketolase molecule manifests itself in the binding of one molecule of the coenzyme.

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.

The role of the charge transfer complex in the transketolase catalyzed reaction

Abstract Thiamine pyrophosphate (TPP), when bound with transketolase (TK) induces some changes in the absorption of the enzyme and coenzyme which can be registered by difference spectrophotometry. The binding of a donor substrate to the binary complex give rise to changes in the absorption region of the TPP thiazolium ring and in the charge transfer spectrum. With low concentrations of hydroxypyruvate, the kinetics of these changes may be revealed. The possibility is discussed of the charge transfer complex (CTC) being involved in the catalytic reaction.

A complex of functionally-bound enzymes: Transketolase and glyceraldehydephosphate dehydrogenase

Abstract By the methods of ion exchange chromatography and disc electrophoresis on polyacrylamide gel it has been shown that in the enzyme preparations isolated from bakers yeast there are both free transketolase and glyceraldehydephospate-dehydrogenase and the complex of these functionally bound enzymes. Being stored in alkaline solution of ammonium sulphate this complex dissociates into the above two components.

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.