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PubMed1977

Solvent effects on thiamin-enzyme model interactions. I. Interactions with tryptophan.

The solvent polarity dependence of the interaction between thiamin and tryptophan was studied by spectrophotometric methods. The ultraviolet (UV) data clearly indicate that the interaction is weakened when the complex is transferred from water to aqueous ethanol or aqueous dioxane. The interaction of thiamin and tryptophan could also be detected by fluorescence-quenching studies (excitation of tryptophan at 287 nm, maximum emission at 348 nm). Appropriate treatment of the quenching data allowed dissection into static and dynamic contributions. A pyrimidine derivative related to thiamin, both in its neutral and protonated states, was shown to interact with tryptophan by fluorescence techniques, but not by UV. A thiazolium model was shown to interact with tryptophan by UV but was an inefficient quencher of the tryptophan fluorescence. Theoretical models are presented to explain the solvent dielectric constant dependence of the association constant between tryptophan and thiamin. Both electrostatic and dispersion forces are found to contribute to the stability of the complex.

Binding Sites↗
PubMed2001

Binding and activation of thiamin diphosphate in acetohydroxyacid synthase.

Acetohydroxyacid synthases (AHASs) are biosynthetic thiamin diphosphate- (ThDP) and FAD-dependent enzymes. They are homologous to pyruvate oxidase and other members of a family of ThDP-dependent enzymes which catalyze reactions in which the first step is decarboxylation of a 2-ketoacid. AHAS catalyzes the condensation of the 2-carbon moiety, derived from the decarboxylation of pyruvate, with a second 2-ketoacid, to form acetolactate or acetohydroxybutyrate. A structural model for AHAS isozyme II (AHAS II) from Escherichia coli has been constructed on the basis of its homology with pyruvate oxidase from Lactobacillus plantarum (LpPOX). We describe here experiments which further test the model, and test whether the binding and activation of ThDP in AHAS involve the same structural elements and mechanism identified for homologous enzymes. Interaction of a conserved glutamate with the N1' of the ThDP aminopyrimidine moiety is involved in activation of the cofactor for proton exchange in several ThDP-dependent enzymes. In accord with this, the analogue N3'-pyridyl thiamin diphosphate does not support AHAS activity. Mutagenesis of Glu47, the putative conserved glutamate, decreases the rate of proton exchange at C-2 of bound ThDP by nearly 2 orders of magnitude and decreases the turnover rate for the mutants by about 10-fold. Mutant E47A also has altered substrate specificity, pH dependence, and other changes in properties. Mutagenesis of Asp428, presumed on the basis of the model to be the crucial carboxylate ligand to Mg(2+) in the "ThDP motif", leads to a decrease in the affinity of AHAS II for Mg(2+). While mutant D428N shows ThDP affinity close to that of the wild-type on saturation with Mg(2+), D428E has a decreased affinity for ThDP. These mutations also lead to dependence of the enzyme on K(+). These experiments demonstrate that AHAS binds and activates ThDP in the same way as do pyruvate decarboxylase, transketolase, and other ThDP-dependent enzymes. The biosynthetic activity of AHAS also involves many other factors beyond the binding and deprotonation of ThDP; changes in the ligands to ThDP can have interesting and unexpected effects on the reaction.

Acetolactate Synthase↗
PubMed2002

Structure of the pyruvate dehydrogenase multienzyme complex E1 component from Escherichia coli at 1.85 A resolution.

The crystal structure of the recombinant thiamin diphosphate-dependent E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) has been determined at a resolution of 1.85 A. The E. coli PDHc E1 component E1p is a homodimeric enzyme and crystallizes with an intact dimer in an asymmetric unit. Each E1p subunit consists of three domains: N-terminal, middle, and C-terminal, with all having alpha/beta folds. The functional dimer contains two catalytic centers located at the interface between subunits. The ThDP cofactors are bound in the "V" conformation in clefts between the two subunits (binding involves the N-terminal and middle domains), and there is a common ThDP binding fold. The cofactors are completely buried, as only the C2 atoms are accessible from solution through the active site clefts. Significant structural differences are observed between individual domains of E1p relative to heterotetrameric multienzyme complex E1 components operating on branched chain substrates. These differences may be responsible for reported alternative E1p binding modes to E2 components within the respective complexes. This paper represents the first structural example of a functional pyruvate dehydrogenase E1p component from any species. It also provides the first representative example for the entire family of homodimeric (alpha2) E1 multienzyme complex components, and should serve as a model for this class of enzymes.

Amino Acid Sequence↗
PubMed2003

Structural and kinetic analysis of catalysis by a thiamin diphosphate-dependent enzyme, benzoylformate decarboxylase.

Benzoylformate decarboxylase is a member of the family of enzymes that are dependent on the cofactor thiamin diphosphate. A structure of this enzyme binding (R)-mandelate, a competitive inhibitor, suggests that at least two hydrogen bonds are formed between the substrate, benzoylformate, and active site side chains. The first is between the carboxylate group of benzoylformate and the hydroxyl group of S26, and the second is between carbonyl group of the substrate and an imidazole nitrogen of H70. Steady-state kinetic studies indicate that the catalytic parameters are strongly affected in three active site mutants, S26A, H70A, and H281A. The K(m) of S26A was increased most dramatically, 25-fold more than that of the wild-type enzyme, while the K(i) of (R)-mandelate was increased 100-fold, suggesting that the serine hydroxyl is important for substrate binding. The k(cat) of H70A is reduced more than 3 orders of magnitude, strongly implicating this residue in catalysis, and H281 showed significant, but smaller magnitude, effects on both K(m) and k(cat). Stopped-flow experiments using an alternative substrate, p-nitrobenzoylformate, lead to kinetic resolution of the fate of key thiamin diphosphate-bound intermediates. Together, the experimental results suggest the following roles for residues in the active site. The residue H70 is important for the protonation of the 2-alpha-mandelyl-ThDP intermediate, thereby assisting in decarboxylation, and for the deprotonation of the 2-alpha-hydroxybenzyl-ThDP intermediate, aiding product release. H281 is involved in protonation of the enamine. Surprisingly, S26 appears to be involved not only in substrate binding but also in other steps of the reaction.

Alanine↗
PubMed2004

Structural determinants of enzyme binding affinity: the E1 component of pyruvate dehydrogenase from Escherichia coli in complex with the inhibitor thiamin thiazolone diphosphate.

Thiamin thiazolone diphosphate (ThTDP), a potent inhibitor of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), binds to the enzyme with greater affinity than does the cofactor thiamin diphosphate (ThDP). To identify what determines this difference, the crystal structure of the apo PDHc E1 component complex with ThTDP and Mg(2+) has been determined at 2.1 A and compared to the known structure of the native holoenzyme, PDHc E1-ThDP-Mg(2+) complex. When ThTDP replaces ThDP, reorganization occurs in the protein structure in the vicinity of the active site involving positional and conformational changes in some amino acid residues, a change in the V coenzyme conformation, addition of new hydration sites, and elimination of others. These changes culminate in an increase in the number of hydrogen bonds to the protein, explaining the greater affinity of the apoenzyme for ThTDP. The observed hydrogen bonding pattern is not an invariant feature of ThDP-dependent enzymes but rather specific to this enzyme since the extra hydrogen bonds are made with nonconserved residues. Accordingly, these sequence-related hydrogen bonding differences likewise explain the wide variation in the affinities of different thiamin-dependent enzymes for ThTDP and ThDP. The sequence of each enzyme determines its ability to form hydrogen bonds to the inhibitor or cofactor. Mechanistic roles are suggested for the aforementioned reorganization and its reversal in PDHc E1 catalysis: to promote substrate binding and product release. This study also provides additional insight into the role of water in enzyme inhibition and catalysis.

Binding Sites↗
PubMed2003

NMR analysis of covalent intermediates in thiamin diphosphate enzymes.

Enzymic catalysis proceeds via intermediates formed in the course of substrate conversion. Here, we directly detect key intermediates in thiamin diphosphate (ThDP)-dependent enzymes during catalysis using (1)H NMR spectroscopy. The quantitative analysis of the relative intermediate concentrations allows the determination of the microscopic rate constants of individual catalytic steps. As demonstrated for pyruvate decarboxylase (PDC), this method, in combination with site-directed mutagenesis, enables the assignment of individual side chains to single steps in catalysis. In PDC, two independent proton relay systems and the stereochemical control of the enzymic environment account for proficient catalysis proceeding via intermediates at carbon 2 of the enzyme-bound cofactor. The application of this method to other ThDP-dependent enzymes provides insight into their specific chemical pathways.

Binding Sites↗
PubMed2003

Steady-state kinetics and molecular evolution of Escherichia coli MenD [(1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase], an anomalous thiamin diphosphate-dependent decarboxylase-carboligase.

(1R,6R)-2-Succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) synthase, or MenD, catalyzes the thiamin diphosphate- (ThDP-) dependent decarboxylation of 2-oxoglutarate, the subsequent addition of the resulting succinyl-ThDP moiety to isochorismate, and the delta-elimination of pyruvate to yield SHCHC, pyruvate, and carbon dioxide. The enzyme is part of a superfamily of ThDP-dependent 2-oxo acid decarboxylases that includes pyruvate decarboxylase, benzoylformate decarboxylase, and acetohydroxy acid synthase, among others. However, this is the only enzyme known to catalyze a Stetter-like 1,4-addition of a ThDP adduct to the beta-carbon of an unsaturated carboxylate. Herein we report properties of the MenD protein from Escherichia coli, including the results of the first steady-state kinetic studies of the SHCHC synthase reaction. The protein is a dimer and shows cooperativity with respect to both substrates. The enzyme prefers divalent manganese as its metal ion cofactor and shows no dependence on FAD. MenD, required for biosynthesis of menaquinone and phylloquinone, is found in the genomes of a wide range of bacteria, as well as that of the archaeon Halobacterium sp. NRC-1 and the eukaryote Arabidopsis thaliana. Sequence alignments with other members of the superfamily are used to predict amino acid residues likely to be important in the binding and activation of ThDP. A site-directed mutant that replaces the conserved glutamic acid residue (E55), predicted to interact with N1' of the aminopyrimidine ring, with glutamine was generated, with catastrophic results for catalysis. There is no evidence for the release of succinate semialdehyde as a product; therefore, EC 4.1.1.71 should not be used for this enzyme.

Amino Acid Sequence↗
PubMed2005

Intermediates and transition states in thiamin diphosphate-dependent decarboxylases. A kinetic and NMR study on wild-type indolepyruvate decarboxylase and variants using indolepyruvate, benzoylformate, and pyruvate as substrates.

The thiamin diphosphate (ThDP)-dependent enzyme indolepyruvate decarboxylase (IPDC) is involved in the biosynthetic pathway of the phytohormone 3-indoleacetic acid and catalyzes the nonoxidative decarboxylation of 3-indolepyruvate to 3-indoleacetaldehyde and carbon dioxide. The steady-state distribution of covalent ThDP intermediates of IPDC reacting with 3-indolepyruvate and the alternative substrates benzoylformate and pyruvate has been analyzed by (1)H NMR spectroscopy. For the first time, we are able to isolate and directly assign covalent intermediates of ThDP with aromatic substrates. The intermediate analysis of IPDC variants is used to infer the involvement of active site side chains and functional groups of the cofactor in distinct catalytic steps during turnover of the different substrates. As a result, three residues (glutamate 468, aspartate 29, and histidine 115) positioned perpendicular to the thiazolium moiety of ThDP are involved in binding of all substrates and decarboxylation of the respective tetrahedral ThDP-substrate adducts. Most likely, interactions of these side chains with the substrate-derived carboxylate account for an optimal orientation of the substrate and/or intermediate in the course of carbon-carbon ligation and decarboxylation supporting the suggested least-motion, maximum overlap mechanism. The active site residue glutamine 383, which is located at the opposite site of the thiazolium nucleus as the "carboxylate pocket" (formed by the Glu-Asp-His triad), is central to the substrate specificity of IPDC, probably through orbital alignment. The Glu51-cofactor proton shuttle is, conjointly with the Glu-Asp-His triad, involved in multiple proton transfer steps, including ylide generation, substrate binding, and product release. Studies with para-substituted benzoylformate substrates demonstrate that the electronic properties of the substrate affect the stabilization or destabilization of the carbanion intermediate or carbanion-like transition state and in that way alter the rate dependence on decarboxylation. In conclusion, general mechanistic principles of catalysis of ThDP-dependent enzymes are discussed.

Amino Acid Substitution↗
PubMed2005

Evidence for dramatic acceleration of a C-H bond ionization rate in thiamin diphosphate enzymes by the protein environment.

The hypothesis that thiamin diphosphate-dependent enzymes achieve a significant fraction of their catalytic rate acceleration by providing a protein environment that helps to stabilize unstable zwitterionic/dipolar intermediates (including the enamine/C2alpha-carbanion present on all such enzymes) was tested experimentally using the intermediate C2alpha-hydroxyethylthiamin diphosphate (HEThDP) with the Escherichia coli pyruvate dehydrogenase complex and its E1 subunit (PDHc-E1). Using pre-steady-state and steady-state methods, it was shown that HEThDP is a substrate for this enzyme after ionization of the C2alpha-H bond. An experiment was then carried out to measure the PDHc-E1 catalyzed pre-steady-state rate constant for the D --> H exchange from the C2alpha position of HEThDP-d(4), as an indicator of the formation of the enamine. Importantly, the enzyme accelerates the rate of ionization of this bond by a factor of 10(7), corresponding to a 10 kcal/mol stabilization of the enamine intermediate by the enzyme. This finding is likely a general feature of thiamin diphosphate enzymes.

Acetylation↗
PubMed2005

Elucidating the specificity of binding of sulfonylurea herbicides to acetohydroxyacid synthase.

Acetohydroxyacid synthase (AHAS, EC 2.2.1.6) is the target for the sulfonylurea herbicides, which act as potent inhibitors of the enzyme. Chlorsulfuron (marketed as Glean) and sulfometuron methyl (marketed as Oust) are two commercially important members of this family of herbicides. Here we report crystal structures of yeast AHAS in complex with chlorsulfuron (at a resolution of 2.19 A), sulfometuron methyl (2.34 A), and two other sulfonylureas, metsulfuron methyl (2.29 A) and tribenuron methyl (2.58 A). The structures observed suggest why these inhibitors have different potencies and provide clues about the differential effects of mutations in the active site tunnel on various inhibitors. In all of the structures, the thiamin diphosphate cofactor is fragmented, possibly as the result of inhibitor binding. In addition to thiamin diphosphate, AHAS requires FAD for activity. Recently, it has been reported that reduction of FAD can occur as a minor side reaction due to reaction with the carbanion/enamine of the hydroxyethyl-ThDP intermediate that is formed midway through the catalytic cycle. Here we report that the isoalloxazine ring has a bent conformation that would account for its ability to accept electrons from the hydroxyethyl intermediate. Most sequence and mutation data suggest that yeast AHAS is a high-quality model for the plant enzyme.

Acetolactate Synthase↗
PubMed2004

Tetrahedral intermediates in thiamin diphosphate-dependent decarboxylations exist as a 1',4'-imino tautomeric form of the coenzyme, unlike the michaelis complex or the free coenzyme.

Two circular dichroism signals observed on thiamin diphosphate (ThDP)-dependent enzymes, a positive band in the 300-305 nm range and a negative one in the 320-330 nm range, were investigated on yeast pyruvate decarboxylase (YPDC) and on the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex (PDHc-E1). Addition of the tetrahedral ThDP-acetaldehyde adduct, 2-alpha-hydroxyethylThDP, to PDHc-E1 generates the positive band at 300 nm, consistent with the formation of the 1',4'-iminopyrimidine tautomer, as also demonstrated for phosphonolactylthiamin diphosphate, a stable analogue of the tetrahedral ThDP-pyruvate adduct 2-alpha-lactylThDP (Jordan, F. et al. (2003) J. Am. Chem. Soc. 125, 12732-12738). Therefore, we suggest that all tetrahedral ThDP-bound covalent complexes will also prefer this tautomer, and that the 4'-aminopyrimidine of ThDP participates in multiple steps of acid-base catalysis on ThDP enzymes. Studies with YPDC and PDHc-E1, and their active center variants, in conjunction with chemical models, enabled assignment of the negative band at 330 nm to a charge-transfer transition between the 4'-aminopyrimidine tautomer (presumed electron donor) and the thiazolium ring (presumed electron acceptor) of ThDP, with no significant contributions from any amino acid side chain of the proteins. However, in both YPDC and PDHc-E1, the presence of substrate or substrate surrogate was required to enable detection, suggesting that the band at 320-330 nm be used as a reporter for the Michaelis complex, involving the amino tautomer, on both enzymes. As the positive band near 300 nm reports on the 1',4'-imino tautomer of ThDP, methods are now available for kinetic monitoring of both tautomeric forms.

Binding Sites↗