Gerhard Hübner

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.

COFACTOR DESIGNING IN FUNCTIONAL ANALYSIS OF THIAMIN DIPHOSPHATE ENZYMES

Publisher Summary Thiamin diphosphate (ThDP) has evolved as a cofactor of enzymes catalyzing the splitting and resynthesis of C–C bonds. It reacts exclusively with substrates of the general structure R–CO–X, where X as a cationic leaving group (CO 2 or R–CHO) is replaced by another positively charged residue (Y). ThDP enzymes are pyruvate decarboxylase (PDC) with Y = H - , transketolases (TK) and acetolactate synthase (ALS), with carbonyl compounds as Y, or the α -oxoacid dehydrogenase complexes, such as pyruvate dehydrogenase complex (PDH), with a cyclic disulfide. In all ThDP enzymes the first substrate contains, besides the activated carbonyl group and the cationic leaving group X, an alkyl residue R, which defines the substrate specificity of the ThDP enzyme. The second substrate Y characterizes the enzyme species. This chapter presents the method of cofactor designing in functional analysis of thiamin diphosphate enzymes. Methods have been developed that describe the binding properties of inactive analogs, helping to elucidate which groups are involved in binding and the specific influence of molecular parameters on the binding mechanism of the cofactors.

The influence of the effectors of yeast pyruvate decarboxylase (PDC) on the conformation of the dimers and tetramers and their pH-dependent equilibrium.

The influence of effectors of yeast pyruvate decarboxylase, phosphate, pyruvamide, thiamin diphosphate and Mg++, on the pH-dependent equilibrium between dimers and tetramers was studied by synchrotron radiation X-ray solution scattering. Thiamin diphosphate and phosphate shift the equilibrium to higher pH values without altering the structure of the oligomers. Pyruvamide, a substrate analogue activator, induces a significant change in the structure of the tetramer. By eliminating radiation damage by addition of dithioerythrol to the buffers, the scattering curves could be measured accurately over a large angular range. They were expanded in terms of spherical harmonics to obtain the shapes of the dimers and tetramers with higher resolution than was hitherto possible. This also allowed us to position the dimers, which are centrosymmetric at low resolution, in the tetramers which have 222 symmetry. The results indicate that addition of pyruvamide results in a less compact tetramer owing to structural changes in the dimers and to their displacements.

Glutamate 636 of the Escherichia coli pyruvate dehydrogenase-E1 participates in active center communication and behaves as an engineered acetolactate synthase with unusual stereoselectivity.

The residue Glu636 is located near the thiamine diphosphate (ThDP) binding site of the Escherichia coli pyruvate dehydrogenase complex E1 subunit (PDHc-E1), and to probe its function two variants, E636A and E636Q were created with specific activities of 2.5 and 26% compared with parental PDHc-E1. According to both fluorescence binding and kinetic assays, the E636A variant behaved according to half-of-the-sites mechanism with respect to ThDP. In contrast, with the E636Q variant a Kd,ThDP = 4.34 μm and Km,ThDP = 11 μm were obtained with behavior more reminiscent of the parental enzyme. The CD spectra of both variants gave evidence for formation of the 1′,4′-iminopyrimidine tautomer on binding of phosphonolactylthiamine diphosphate, a stable analog of the substrate-ThDP covalent complex. Rapid formation of optically active (R)-acetolactate by both variants, but not by the parental enzyme, was observed by CD and NMR spectroscopy. The acetolactate configuration produced by the Glu636 variants is opposite that produced by the enzyme acetolactate synthase and the Asp28-substituted variants of yeast pyruvate decarboxylase, suggesting that the active centers of the two sets of enzymes exhibit different facial selectivity (re or si) vis à vis pyruvate. The tryptic peptide map (mass spectral analysis) revealed that the Glu636 substitution changed the mobility of a loop comprising amino acid residues from the ThDP binding fold. Apparently, the residue Glu636 has important functions both in active center communication and in protecting the active center from undesirable “carboligase” side reactions.

Activation of thiamin diphosphate and FAD in the phosphatedependent pyruvate oxidase from Lactobacillus plantarum.

The phosphate- and oxygen-dependent pyruvate oxidase from Lactobacillus plantarum is a homotetrameric enzyme that binds 1 FAD and 1 thiamine diphosphate per subunit. A kinetic analysis of the partial reactions in the overall oxidative conversion of pyruvate to acetyl phosphate and CO2 shows an indirect activation of the thiamine diphosphate by FAD that is mediated by the protein moiety. The rate constant of the initial step, the deprotonation of C2-H of thiamine diphosphate, increases 10-fold in the binary apoenzyme-thiamine diphosphate complex to 10−2 s−1. Acceleration of this step beyond the observed overall catalytic rate constant to 20 s−1 requires enzyme-bound FAD. FAD appears to bind in a two-step mechanism. The primarily bound form allows formation of hydroxyethylthiamine diphosphate but not the transfer of electrons from this intermediate to O2. This intermediate form can be mimicked using 5-deaza-FAD, which is inactive toward O2 but active in an assay using 2,6-dichlorophenolindophenol as electron acceptor. This analogue also promotes the rate constant of C2-H dissociation of thiamine diphosphate in pyruvate oxidase beyond the overall enzyme turnover. Formation of the catalytically competent FAD-thiamine-pyruvate oxidase ternary complex requires a second step, which was detected at low temperature.

The crystal structure of pyruvate decarboxylase from Kluyveromyces lactis : Implications for the substrate activation mechanism of this enzyme

The crystal structure of pyruvate decarboxylase from Kluyveromyces lactis has been determined to 2.26 Å resolution. Like other yeast enzymes, Kluyveromyces lactis pyruvate decarboxylase is subject to allosteric substrate activation. Binding of substrate at a regulatory site induces catalytic activity. This process is accompanied by conformational changes and subunit rearrangements. In the nonactivated form of the corresponding enzyme from Saccharomyces cerevisiae, all active sites are solvent accessible due to the high flexibility of loop regions 106–113 and 292–301. The binding of the activator pyruvamide arrests these loops. Consequently, two of four active sites become closed. In Kluyveromyces lactis pyruvate decarboxylase, this half‐side closed tetramer is present even without any activator. However, one of the loops (residues 105–113), which are flexible in nonactivated Saccharomyces cerevisiae pyruvate decarboxylase, remains flexible. Even though the tetramer assemblies of both enzyme species are different in the absence of activating agents, their substrate activation kinetics are similar. This implies an equilibrium between the open and the half‐side closed state of yeast pyruvate decarboxylase tetramers. The completely open enzyme state is favoured for Saccharomyces cerevisiae pyruvate decarboxylase, whereas the half‐side closed form is predominant for Kluyveromyces lactis pyruvate decarboxylase. Consequently, the structuring of the flexible loop region 105–113 seems to be the crucial step during the substrate activation process of Kluyveromyces lactis pyruvate decarboxylase.

An X-ray solution scattering study of the cofactor and activator induced structural changes in yeast pyruvate decarboxylase (PDC)

Structure and activation pattern of pyruvate decarboxylase (PDC) from yeast was studied by synchrotron radiation X‐ray solution scattering. The results give a direct proof that the reversible deactivation of PDC at pH 8.0 is accompanied by the dissociation of the tetrameric holoenzyme into dimeric halves. The kinetics of this process was followed. At pH 6.5 the dimeric halves reassociate to a tetramer even in the absence of cofactors. The changes of the scattering pattern upon binding of the substrate‐like activator pyruvamide indicate that the structure expands in the course of the enzyme activation.

Amino Acids Allosterically Regulate the Thiamine Diphosphate-dependent α-Keto Acid Decarboxylase from Mycobacterium tuberculosis

The gene rv0853c from Mycobacterium tuberculosis strain H37Rv codes for a thiamine diphosphate-dependent α-keto acid decarboxylase (MtKDC), an enzyme involved in the amino acid degradation via the Ehrlich pathway. Steady state kinetic experiments were performed to determine the substrate specificity of MtKDC. The mycobacterial enzyme was found to convert a broad spectrum of branched-chain and aromatic α-keto acids. Stopped-flow kinetics showed that MtKDC is allosterically activated by α-keto acids. Even more, we demonstrate that also amino acids are potent activators of this thiamine diphosphate-dependent enzyme. Thus, metabolic flow through the Ehrlich pathway can be directly regulated at the decarboxylation step. The influence of amino acids on MtKDC catalysis was investigated, and implications for other thiamine diphosphate-dependent enzymes are discussed.

The Unique Hexokinase of Kluyveromyces lactis MOLECULAR AND FUNCTIONAL CHARACTERIZATION AND EVALUATION OF A ROLE IN GLUCOSE SIGNALING

The Crabtree-negative yeast Kluyveromyces lactis is capable of adjusting its glycolytic flux to the requirements of respiration by tightly regulating glucose uptake. RAG5 encoding the only glucose and fructose phosphorylating enzyme present in K. lactis is required for the up-regulation of glucose transport and also for glucose repression. To understand the significance of the molecular identity and specific function(s) of the corresponding kinase to glucose signaling, RAG5 was overexpressed and its gene product KlHxk1 (Rag5p) isolated and characterized. Stopped-flow kinetics and sedimentation analysis indicated a monomer-homodimer equilibrium of KlHxk1 in a condition of catalysis, i.e. in the presence of substrates and products. The kinetic constants of ATP-dependent glucose phosphorylation identified a 53-kDa monomer as the high affinity/high activity form of the novel enzyme for both glycolytic substrates suggesting a control of glucose phosphorylation at the level of dimer formation and dissociation. In contrast to the highly homologous hexokinase isoenzyme 2 of Saccharomyces cerevisiae (ScHxk2), KlHxk1 was not inhibited by free ATP in a physiological range of nucleotide concentration. Mass spectrometric sequencing of tryptic peptides of KlHxk1 identified unmodified serine at amino acid position 156. The corresponding amino acid in ScHxk2 is serine 157, which represents the autophosphorylation-inactivation site. KlHxk1 did not display, however, the typical pattern of inactivation under the respective in vitro conditions and maintained a high residual glucose phosphorylating activity. The biophysical and functional data are discussed with respect to a possible regulatory role of KlHxk1 in glucose metabolism and signaling in K. lactis.

Activation of thiamine diphosphate in pyruvate decarboxylase from Zymomonas mobilis

Replacement of tryptophan 392 located in the active site cavity of pyruvate decarboxylase (PDC; EC 4.1.1.1) from Zymomonas mobilis by methionine or glutamine yields enzymes with smaller catalytic constants of 8.5 s−1 and 3.6 s−1 at 4°C, compared to that of the wild‐type enzyme (17 s−1). The rate constants of the H/D exchange at the C2 of the coenzyme thiamine diphosphate have been determined to be 130 s−1 for the wild‐type enzyme, 56 s−1 for the methionine and 30 s−1 for the glutamine mutant, respectively. A group with a pK a of about 5 has been identified to be essential for C2 deprotonation of the enzyme‐bound thiamine diphosphate from the pH dependence of the H/D exchange.