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.     

A dynamic loop at the active center of the Escherichia coli pyruvate dehydrogenase complex E1 component modulates substrate utilization and chemical communication with the E2 component

Our crystallographic studies have shown that two active center loops (an inner loop formed by residues 401-413 and outer loop formed by residues 541-557) of the E1 component of the Escherichia coli pyruvate dehydrogenase complex become organized only on binding a substrate analog that is capable of forming a stable thiamin diphosphate-bound covalent intermediate. We showed that residue His-407 on the inner loop has a key role in the mechanism, especially in the reductive acetylation of the E. coli dihydrolipoamide transacetylase component, whereas crystallographic results showed a role of this residue in a disorder-order transformation of these two loops, and the ordered conformation gives rise to numerous new contacts between the inner loop and the active center. We present mapping of the conserved residues on the inner loop. Kinetic, spectroscopic, and crystallographic studies on some inner loop variants led us to conclude that charged residues flanking His-407 are important for stabilization/ordering of the inner loop thereby facilitating completion of the active site. The results further suggest that a disorder to order transition of the dynamic inner loop is essential for substrate entry to the active site, for sequestering active site chemistry from undesirable side reactions, as well as for communication between the E1 and E2 components of the E. coli pyruvate dehydrogenase multienzyme complex.     

Multiple roles of mobile active center loops in the E1 component of the Escherichia coli pyruvate dehydrogenase complex—Linkage of protein dynamics to catalysis

The region encompassing residues 401–413 on the E1 component of the pyruvate dehydrogenase multienzyme complex from Escherichia coli comprises a loop (the inner loop) which was not seen in the X-ray structure in the presence of thiamin diphosphate, the required cofactor for the enzyme. This loop is seen in the presence of a stable analogue of the pre-decarboxylation intermediate, the covalent adduct between the substrate analogue methyl acetylphosphonate and thiamin diphosphate, C2α-phosphonolactylthiamin diphosphate. It has been shown that the residue H407 and several other residues on this loop are required to reduce the mobility of the loop so electron density corresponding to it can be seen once the pre-decarboxylation intermediate is formed. Concomitantly, the loop encompassing residues 541–557 (the outer loop) appears to work in tandem with the inner loop and there is a hydrogen bond between the two loops ensuring their correlated motion. The inner loop was shown to: (a) sequester the active center from carboligase side reactions; (b) assist the interaction between the E1 and the E2 components, thereby affecting the overall reaction rate of the entire multienzyme complex; (c) control substrate access to the active center. Using viscosity effects on kinetics it was shown that formation of the pre-decarboxylation intermediate is specifically affected by loop movement. A cysteine-less variant was created for the E1 component, onto which cysteines were substituted at selected loop positions. Introducing an electron spin resonance spin label and an ¹⁹F NMR label onto these engineered cysteines, the loop mobility was examined: (a) both methods suggested that in the absence of ligand, the loop exists in two conformations; (b) line-shape analysis of the NMR signal at different temperatures, enabled estimation of the rate constant for loop movement, and this rate constant was found to be of the same order of magnitude as the turnover number for the enzyme under the same conditions. Furthermore, this analysis gave important insights into rate-limiting thermal loop dynamics. Overall, the results suggest that the dynamic properties correlate with catalytic events on the E1 component of the pyruvate dehydrogenase complex.     

Remarkable stabilization of zwitterionic intermediates may account for a billion-fold rate acceleration by thiamin diphosphate-dependent decarboxylases.

When the E91D variant of apo-yeast pyruvate decarboxylase (EC 4.1.1. 1) is exposed to C2alpha-hydroxybenzylthiamin diphosphate, this putative intermediate is partitioned on the enzyme between release of the benzaldehyde product (as evidenced by regeneration of active enzyme) and dissociation of the proton at C2alpha to form the enamine-C2alpha-carbanion intermediate. While the pKa (the negative log of the acid dissociation constant) for this dissociation is approximately 15.4 in water, formation of the enamine at pH 6.0 on the enzyme indicates a >9 unit pKa suppression by the enzyme environment. The dramatic stabilization of this zwitterionic enamine intermediate at the active center is sufficient to account for as much as a 10(9)-fold rate acceleration on the enzyme. This "solvent" effect could be useful for achieving the bulk of the rate acceleration provided by the protein over and above that afforded by the coenzyme on all thiamin diphosphate-dependent 2-oxo acid decarboxylases.     

Direct kinetic evidence for half-of-the-sites reactivity in the E1 component of the human pyruvate dehydrogenase multienzyme complex through alternating sites cofactor activation

Recent kinetic and structural studies on various thiamin-dependent enzymes, including the bacterial E1 component of the pyruvate dehydrogenase complex (PDHc), suggested an active center communication between the cofactors in these multimeric enzymes. This regulatory mode has been inferred from the dissymmetry of active sites in proteolytic patterns and X-ray structures and from a complex macroscopic kinetic behavior not being consistent with independently working active sites. Here, direct microscopic kinetic evidence for this hypothesis is presented for the alpha2beta2-type E1 component of the human pyruvate dehydrogenase complex. Only one of the two thiamin molecules bound to the two active sites is in a chemically activated state exhibiting an apparent C2 ionization rate constant of approximately 50 s(-1) at pH 7.6 and 30 degrees C, whereas the thiamin in the "inactive site" ionizes with a rate that is at least 3 orders of magnitude smaller. The chemical nonequivalence is also exhibited in the ability to bind the substrate analogue methyl acetylphosphonate and in the catalytic turnover of the substrate pyruvate in the E1-only reaction. In the activated active site, pyruvate is rapidly bound and decarboxylated with apparent forward rate constants of covalent pyruvate binding of 2 s(-1) and decarboxylation of the formed 2-lactyl-thiamin intermediate of 5 s(-1). In the dormant site, these steps are as slow as 0.03 s(-1). Under the conditions that were used, only the heterotetramer can be detected by analytical ultracentrifugation, thus ruling out the possibility that multiple oligomeric species with different reactivities cause the observed kinetic effects. The results are consistent with the recently suggested model of an active site synchronization in PDHc-E1 via a proton wire that keeps the two active sites in an alternating activation state [Frank, R. A., et al. (2004) Science 306, 872]. Kinetic studies on the related thiamin enzymes transketolase, pyruvate oxidase, and bacterial pyruvate decarboxylase are not consistent with a chemical and/or functional nonequivalence of the active sites as observed in the E1 component of hsPDHc. We hypothesize that the alternating sites reaction in PDHc-E1 aids in the synchronized acyl transfer to the E2 component in the highly organized multienzyme complex.     

Crystal structure of thiamindiphosphate-dependent indolepyruvate decarboxylase from Enterobacter cloacae, an enzyme involved in the biosynthesis of the plant hormone indole-3-acetic acid.

The thiamin diphosphate-dependent enzyme indolepyruvate decarboxylase catalyses the formation of indoleacetaldehyde from indolepyruvate, one step in the indolepyruvate pathway of biosynthesis of the plant hormone indole-3-acetic acid. The crystal structure of this enzyme from Enterobacter cloacae has been determined at 2.65 A resolution and refined to a crystallographic R-factor of 20.5% (Rfree 23.6%). The subunit of indolepyruvate decarboxylase contains three domains of open alpha/beta topology, which are similar in structure to that of pyruvate decarboxylase. The tetramer has pseudo 222 symmetry and can be described as a dimer of dimers. It resembles the tetramer of pyruvate decarboxylase from Zymomonas mobilis, but with a relative difference of 20 degrees in the angle between the two dimers. Active site residues are highly conserved in indolepyruvate/pyruvate decarboxylase, suggesting that the interactions with the cofactor thiamin diphosphate and the catalytic mechanisms are very similar. The substrate binding site in indolepyruvate decarboxylase contains a large hydrophobic pocket which can accommodate the bulky indole moiety of the substrate. In pyruvate decarboxylases this pocket is smaller in size and allows discrimination of larger vs. smaller substrates. In most pyruvate decarboxylases, restriction of cavity size is due to replacement of residues at three positions by large, hydrophobic amino acids such as tyrosine or tryptophan.     

Substrate specificity in thiamin diphosphate-dependent decarboxylases

Thiamin diphosphate (ThDP) is the biologically active form of vitamin B₁, and ThDP-dependent enzymes are found in all forms of life. The catalytic mechanism of this family requires the formation of a common intermediate, the 2α-carbanion–enamine, regardless of whether the enzyme is involved in C–C bond formation or breakdown, or even formation of C−N, C−O and C−S bonds. This demands that the enzymes must screen substrates prior to, and/or after, formation of the common intermediate. This review is focused on the group for which the second step is the protonation of the 2α-carbanion, i.e., the ThDP-dependent decarboxylases. Based on kinetic data, sequence/structure alignments and mutagenesis studies the factors involved in substrate specificity have been identified.     

Structure and Function of the Catalytic Domain of the Dihydrolipoyl Acetyltransferase Component in Escherichia coli Pyruvate Dehydrogenase Complex

The Escherichia coli pyruvate dehydrogenase complex (PDHc) catalyzing conversion of pyruvate to acetyl-CoA comprises three components: E1p, E2p, and E3. The E2p is the five-domain core component, consisting of three tandem lipoyl domains (LDs), a peripheral subunit binding domain (PSBD), and a catalytic domain (E2pCD). Herein are reported the following. 1) The x-ray structure of E2pCD revealed both intra- and intertrimer interactions, similar to those reported for other E2pCDs. 2) Reconstitution of recombinant LD and E2pCD with E1p and E3p into PDHc could maintain at least 6.4% activity (NADH production), confirming the functional competence of the E2pCD and active center coupling among E1p, LD, E2pCD, and E3 even in the absence of PSBD and of a covalent link between domains within E2p. 3) Direct acetyl transfer between LD and coenzyme A catalyzed by E2pCD was observed with a rate constant of 199 s⁻¹, comparable with the rate of NADH production in the PDHc reaction. Hence, neither reductive acetylation of E2p nor acetyl transfer within E2p is rate-limiting. 4) An unprecedented finding is that although no interaction could be detected between E1p and E2pCD by itself, a domain-induced interaction was identified on E1p active centers upon assembly with E2p and C-terminally truncated E2p proteins by hydrogen/deuterium exchange mass spectrometry. The inclusion of each additional domain of E2p strengthened the interaction with E1p, and the interaction was strongest with intact E2p. E2p domain-induced changes at the E1p active site were also manifested by the appearance of a circular dichroism band characteristic of the canonical 4′-aminopyrimidine tautomer of bound thiamin diphosphate (AP).     

Elementary steps in the reaction of the pyruvate dehydrogenase complex from pig heart. Kinetics of thiamine diphosphate binding to the complex

In the progress curve of the reaction of the pyruvate dehydrogenase complex, a lag phase was observed when the concentration of thiamin diphosphate was lower than usual (about 0.2-1 mM) in the enzyme assay. The length of the lag phase was dependent on thiamin diphosphate concentration, ranging from 0.2 min to 2 min as the thiamin diphosphate concentration varied from 800 nM to 22 nM. The lag phase was also observed in the elementary steps catalyzed by the pyruvate dehydrogenase component. A Km value of 107 nM was found for thiamin diphosphate with respect to the steady-state reaction rate following the lag phase. The pre-steady-state kinetic data indicate that the resulting lag phase was the consequence of a slow holoenzyme formation from apoenzyme and thiamin diphosphate. The thiamin diphosphate can bind to the pyruvate dehydrogenase complex in the absence of pyruvate, but the presence of 2 mM pyruvate increases the rate constant of binding from 1.4 X 10(4) M-1 S-1 to 1.3 X 10(5) M-1 S-1 and decreases the rate constant of dissociation from 2.3 X 10(-2) S-1 to 4.1 X 10(-3) S-1. On the other hand, the effect of pyruvate on the thiamin diphosphate binding revealed the existence of a thiamin-diphosphate-independent pyruvate-binding site in the pyruvate dehydrogenase complex. Direct evidence was also obtained with fluorescence techniques for the existence of this binding site and the dissociation constant of pyruvate was found to be 0.38 mM. On the basis of these data we have proposed a random mechanism for the binding of pyruvate and thiamin diphosphate to the complex. Binding of substrates to the enzyme complex caused an increase in the fluorescence of the dansylaziridine-labelled pyruvate dehydrogenase complex, showing that binding of substrates to the complex is accompanied by structural changes.     

Synthesis with good enantiomeric excess of both enantiomers of alpha-ketols and acetolactates by two thiamin diphosphate-dependent decarboxylases

In addition to the decarboxylation of 2-oxo acids, thiamin diphosphate (ThDP)-dependent decarboxylases/dehydrogenases can also carry out so-called carboligation reactions, where the central ThDP-bound enamine intermediate reacts with electrophilic substrates. For example, the enzyme yeast pyruvate decarboxylase (YPDC, from Saccharomyces cerevisiae) or the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex (PDHc-E1) can produce acetoin and acetolactate, resulting from the reaction of the central thiamin diphosphate-bound enamine with acetaldehyde and pyruvate, respectively. Earlier, we had shown that some active center variants indeed prefer such a carboligase pathway to the usual one [Sergienko, Jordan, Biochemistry 40 (2001) 7369-7381; Nemeria et al., J. Biol. Chem. 280 (2005) 21,473-21,482]. Herein is reported detailed analysis of the stereoselectivity for forming the carboligase products acetoin, acetolactate, and phenylacetylcarbinol by the E477Q and D28A YPDC, and the E636A and E636Q PDHc-E1 active-center variants. Both pyruvate and beta-hydroxypyruvate were used as substrates and the enantiomeric excess was analyzed by a combination of NMR, circular dichroism and chiral-column gas chromatographic methods. Remarkably, the two enzymes produced a high enantiomeric excess of the opposite enantiomer of both acetoin-derived and acetolactate-derived products, strongly suggesting that the facial selectivity for the electrophile in the carboligation is different in the two enzymes. The different stereoselectivities exhibited by the two enzymes could be utilized in the chiral synthesis of important intermediates.     

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.     

Probing the active center of benzaldehyde lyase with substitutions and the pseudosubstrate analogue benzoylphosphonic acid methyl ester

Benzaldehyde lyase (BAL) catalyzes the reversible cleavage of ( R)-benzoin to benzaldehyde utilizing thiamin diphosphate and Mg (2+) as cofactors. The enzyme is important for the chemoenzymatic synthesis of a wide range of compounds via its carboligation reaction mechanism. In addition to its principal functions, BAL can slowly decarboxylate aromatic amino acids such as benzoylformic acid. It is also intriguing mechanistically due to the paucity of acid-base residues at the active center that can participate in proton transfer steps thought to be necessary for these types of reactions. Here methyl benzoylphosphonate, an excellent electrostatic analogue of benzoylformic acid, is used to probe the mechanism of benzaldehyde lyase. The structure of benzaldehyde lyase in its covalent complex with methyl benzoylphosphonate was determined to 2.49 A (Protein Data Bank entry 3D7K ) and represents the first structure of this enzyme with a compound bound in the active site. No large structural reorganization was detected compared to the complex of the enzyme with thiamin diphosphate. The configuration of the predecarboxylation thiamin-bound intermediate was clarified by the structure. Both spectroscopic and X-ray structural studies are consistent with inhibition resulting from the binding of MBP to the thiamin diphosphate in the active centers. We also delineated the role of His29 (the sole potential acid-base catalyst in the active site other than the highly conserved Glu50) and Trp163 in cofactor activation and catalysis by benzaldehyde lyase.     

Regulatory properties of the pyruvate dehydrogenase complex from Escherichia coli. Studies on the thiamin diphosphate-dependent lag phase

The pyruvate dehydrogenase complex from Escherichia coli shows an appreciable lag phase (tau) of some minutes when its overall reaction rate was tested with very limiting amounts of thiamin diphosphate. tau depends on the concentration of thiamin diphosphate in a nonlinear fashion. Sodium diphosphate, a competitive inhibitor with respect to thiamin diphosphate (Ki = 5.2 . 10(-4) M) prolongs the lag, while the strongly binding transition state analog thiamin thiazolone diphosphate has no effect. tau is independent of the enzyme concentration, thus no dissociation-association step is involved. Incubation of the pyruvate dehydrogenase complex with thiamin diphosphate, Mg2+, and pyruvate leads to a shortening of the lag phase, as well as to a decrease of the intrinsic tryptophan fluorescence in a time-dependent process, which evinces the same characteristics as tau. Dependence of pyruvate, as well as of the substrate analog methylacetylphosphonate, can be established by measurements of fluorescence quenching, thus ruling out an essential role of hydroxyethyl thiamin diphosphate in the process reflected by the lag phase. The results demonstrate that the lag phase is induced after the binding of both thiamin diphosphate . Mg2+ and pyruvate to the catalytic site to form a ternary enzyme complex, which undergoes subsequently a slow conformational change to an active enzyme form. This change is confined to single subunits, and no interactions between neighboring monomers could be observed. A model is proposed to describe the mechanism represented by the lag phase.     

Synthesis with good enantiomeric excess of both enantiomers of α-ketols and acetolactates by two thiamin diphosphate-dependent decarboxylases

In addition to the decarboxylation of 2-oxo acids, thiamin diphosphate (ThDP)-dependent decarboxylases/dehydrogenases can also carry out so-called carboligation reactions, where the central ThDP-bound enamine intermediate reacts with electrophilic substrates. For example, the enzyme yeast pyruvate decarboxylase (YPDC, from Saccharomyces cerevisiae) or the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex (PDHc-E1) can produce acetoin and acetolactate, resulting from the reaction of the central thiamin diphosphate-bound enamine with acetaldehyde and pyruvate, respectively. Earlier, we had shown that some active center variants indeed prefer such a carboligase pathway to the usual one [Sergienko, Jordan, Biochemistry 40 (2001) 7369–7381; Nemeria et al., J. Biol. Chem. 280 (2005) 21,473–21,482]. Herein is reported detailed analysis of the stereoselectivity for forming the carboligase products acetoin, acetolactate, and phenylacetylcarbinol by the E477Q and D28A YPDC, and the E636A and E636Q PDHc-E1 active-center variants. Both pyruvate and β-hydroxypyruvate were used as substrates and the enantiomeric excess was analyzed by a combination of NMR, circular dichroism and chiral-column gas chromatographic methods. Remarkably, the two enzymes produced a high enantiomeric excess of the opposite enantiomer of both acetoin-derived and acetolactate-derived products, strongly suggesting that the facial selectivity for the electrophile in the carboligation is different in the two enzymes. The different stereoselectivities exhibited by the two enzymes could be utilized in the chiral synthesis of important intermediates.     

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.     

The crystal structures of Klebsiella pneumoniae acetolactate synthase with enzyme-bound cofactor and with an unusual intermediate

Acetohydroxyacid synthase (AHAS) and acetolactate synthase (ALS) are thiamine diphosphate (ThDP)-dependent enzymes that catalyze the decarboxylation of pyruvate to give a cofactor-bound hydroxyethyl group, which is transferred to a second molecule of pyruvate to give 2-acetolactate. AHAS is found in plants, fungi, and bacteria, is involved in the biosynthesis of the branched-chain amino acids, and contains non-catalytic FAD. ALS is found only in some bacteria, is a catabolic enzyme required for the butanediol fermentation, and does not contain FAD. Here we report the 2.3-A crystal structure of Klebsiella pneumoniae ALS. The overall structure is similar to AHAS except for a groove that accommodates FAD in AHAS, which is filled with amino acid side chains in ALS. The ThDP cofactor has an unusual conformation that is unprecedented among the 26 known three-dimensional structures of nine ThDP-dependent enzymes, including AHAS. This conformation suggests a novel mechanism for ALS. A second structure, at 2.0 A, is described in which the enzyme is trapped halfway through the catalytic cycle so that it contains the hydroxyethyl intermediate bound to ThDP. The cofactor has a tricyclic structure that has not been observed previously in any ThDP-dependent enzyme, although similar structures are well known for free thiamine. This structure is consistent with our proposed mechanism and probably results from an intramolecular proton transfer within a tricyclic carbanion that is the true reaction intermediate. Modeling of the second molecule of pyruvate into the active site of the enzyme with the bound intermediate is consistent with the stereochemistry and specificity of ALS.     

Phosphorylation of serine 264 impedes active site accessibility in the E1 component of the human pyruvate dehydrogenase multienzyme complex

At the junction of glycolysis and the Krebs cycle in cellular metabolism, the pyruvate dehydrogenase multienzyme complex (PDHc) catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA. In mammals, PDHc is tightly regulated by phosphorylation-dephosphorylation of three serine residues in the thiamin-dependent pyruvate dehydrogenase (E1) component. In vivo, inactivation of human PDHc correlates mostly with phosphorylation of serine 264, which is located at the entrance of the substrate channel leading to the active site of E1. Despite intense investigations, the molecular mechanism of this inactivation has remained enigmatic. Here, a detailed analysis of microscopic steps of catalysis in human wild-type PDHc-E1 and pseudophosphorylation variant Ser264Glu elucidates how phosphorylation of Ser264 affects catalysis. Whereas the intrinsic reactivity of the active site in catalysis of pyruvate decarboxylation remains nearly unaltered, the preceding binding of substrate to the enzyme's active site via the substrate channel and the subsequent reductive acetylation of the E2 component are severely slowed in the phosphorylation variant. The structure of pseudophosphorylation variant Ser264Glu determined by X-ray crystallography reveals no differences in the three-dimensional architecture of the phosphorylation loop or of the active site, when compared to those of the wild-type enzyme. However, the channel leading to the active site is partially obstructed by the side chain of residue 264 in the variant. By analogy, a similar obstruction of the substrate channel can be anticipated to result from a phosphorylation of Ser264. The kinetic and thermodynamic results in conjunction with the structure of Ser264Glu suggest that phosphorylation blocks access to the active site by imposing a steric and electrostatic barrier for substrate binding and active site coupling with the E2 component. As a Ser264Gln variant, which carries no charge at position 264, is also selectively deficient in pyruvate binding and reductive acetylation of E2, we conclude that mostly steric effects account for inhibition of PDHc by phosphorylation.     

Effect of coenzyme modification on the structural and catalytic properties of wild-type transketolase and of the variant E418A from Saccharomyces cerevisiae

Transketolase from baker's yeast is a thiamin diphosphate-dependent enzyme in sugar metabolism that reconstitutes with various analogues of the coenzyme. The methylated analogues (4'-methylamino-thiamin diphosphate and N1'-methylated thiamin diphosphate) of the native cofactor were used to investigate the function of the aminopyrimidine moiety of the coenzyme in transketolase catalysis. For the wild-type transketolase complex with the 4'-methylamino analogue, no electron density was found for the methyl group in the X-ray structure, whereas in the complex with the N1'-methylated coenzyme the entire aminopyrimidine ring was disordered. This indicates a high flexibility of the respective parts of the enzyme-bound thiamin diphosphate analogues. In the E418A variant of transketolase reconstituted with N1'-methylated thiamin diphosphate, the electron density of the analogue was well defined and showed the typical V-conformation found in the wild-type holoenzyme [Lindqvist Y, Schneider G, Ermler U, Sundstrom M (1992) EMBO J11, 2373-2379]. The near-UV CD spectrum of the variant E418A reconstituted with N1'-methylated thiamin diphosphate was identical to that of the wild-type holoenzyme, while the CD spectrum of the variant combined with the unmodified cofactor did not overlap with that of the native protein. The activation of the analogues was measured by the H/D-exchange at C2. Methylation at the N1' position of the cofactor activated the enzyme-bound cofactor analogue (as shown by a fast H/D-exchange rate constant). The absorbance changes in the course of substrate turnover of the different complexes investigated (transient kinetics) revealed the stability of the alpha-carbanion/enamine as the key intermediate in cofactor action to be dependent on the functionality of the 4-aminopyrimidine moiety of thiamin diphosphate.     

The two active sites in human branched-chain alpha-keto acid dehydrogenase operate independently without an obligatory alternating-site mechanism

A long standing controversy is whether an alternating activesite mechanism occurs during catalysis in thiamine diphosphate (ThDP)-dependent enzymes. We address this question by investigating the ThDP-dependent decarboxylase/dehydrogenase (E1b) component of the mitochondrial branched-chain alpha-keto acid dehydrogenase complex (BCKDC). Our crystal structure reveals that conformations of the two active sites in the human E1b heterotetramer harboring the reaction intermediate are identical. Acidic residues in the core of the E1b heterotetramer, which align with the proton-wire residues proposed to participate in active-site communication in the related pyruvate dehydrogenase from Bacillus stearothermophilus, are mutated. Enzyme kinetic data show that, except in a few cases because of protein misfolding, these alterations are largely without effect on overall activity of BCKDC, ruling out the requirement of a proton-relay mechanism in E1b. BCKDC overall activity is nullified at 50% phosphorylation of E1b, but it is restored to nearly half of the pre-phosphorylation level after dissociation and reconstitution of BCKDC with the same phosphorylated E1b. The results suggest that the abolition of overall activity likely results from the specific geometry of the half-phosphorylated E1b in the BCKDC assembly and not due to a disruption of the alternating active-site mechanism. Finally, we show that a mutant E1b containing only one functional active site exhibits half of the wild-type BCKDC activity, which directly argues against the obligatory communication between active sites. The above results provide evidence that the two active sites in the E1b heterotetramer operate independently during the ThDP-dependent decarboxylation reaction.     

Site-directed mutagenesis of a loop at the active site of E1 (alpha2beta2) of the pyruvate dehydrogenase complex. A possible common sequence motif.

Limited proteolysis of the pyruvate decarboxylase (E1, alpha2beta2) component of the pyruvate dehydrogenase (PDH) multienzyme complex of Bacillus stearothermophilus has indicated the importance for catalysis of a site (Tyr281-Arg282) in the E1alpha subunit (Chauhan, H.J., Domingo, G.J., Jung, H.-I. & Perham, R.N. (2000) Eur. J. Biochem. 267, 7158-7169). This site appears to be conserved in the alpha-subunit of heterotetrameric E1s and multiple sequence alignments suggest that there are additional conserved amino-acid residues in this region, part of a common pattern with the consensus sequence -YR-H-D-YR-DE-. This region lies about 50 amino acids on the C-terminal side of a 30-residue motif previously recognized as involved in binding thiamin diphosphate (ThDP) in all ThDP-dependent enzymes. The role of individual residues in this set of conserved amino acids in the E1alpha chain was investigated by means of site-directed mutagenesis. We propose that particular residues are involved in: (a) binding the 2-oxo acid substrate, (b) decarboxylation of the 2-oxo acid and reductive acetylation of the tethered lipoyl domain in the PDH complex, (c) an "open-close" mechanism of the active site, and (d) phosphorylation by the E1-specific kinase (in eukaryotic PDH and branched chain 2-oxo acid dehydrogenase complexes).