Active-site modification of mammalian pyruvate dehydrogenase by pyridoxal 5'-phosphate

The pyruvate dehydrogenase multienzyme complex from bovine kidney and heart is inactivated by treatment with pyridoxal 5'-phosphate and sodium cyanide or sodium borohydride. The site of this inhibition is the pyruvate dehydrogenase (E1) component of the complex. Inactivation of E1 by the pyridoxal phosphate-cyanide treatment was prevented by thiamin pyrophosphate. Equilibrium binding studies showed that E1 contains two thiamin pyrophosphate binding sites per molecule (alpha 2 beta 2) and that modification of E1 increased the dissociation constant (Kd) for thiamin pyrophosphate about 5-fold. Incorporation of approximately 2.4 equiv of 14CN per mole of E1 tetramer in the presence of pyridoxal phosphate resulted in about a 90% loss of E1 activity. Radioactivity was incorporated predominantly into the E1 alpha subunit. Radioactive N6-pyridoxyllysine was identified in an acid hydrolysate of the E1-pyridoxal phosphate complex that had been reduced with NaB3H4. The data are interpreted to indicate that in the presence of sodium cyanide or sodium borohydride, pyridoxal phosphate reacts with a lysine residue at or near the thiamin pyrophosphate binding site of E1. This binding site is apparently located on the alpha subunit.     

The elementary reactions of the pig heart pyruvate dehydrogenase complex. A study of the inhibition by phosphorylation.

1. A method was devised for preparing pig heart pyruvate dehydrogenase free of thiamin pyrophosphate (TPP), permitting studies of the binding of [35S]TPP to pyruvate dehydrogenase and pyruvate dehydrogenase phosphate. The Kd of TPP for pyruvate dehydrogenase was in the range 6.2-8.2 muM, whereas that for pyruvate dehydrogenase phosphate was approximately 15 muM; both forms of the complex contained about the same total number of binding sites (500 pmol/unit of enzyme). EDTA completely inhibited binding of TPP; sodium pyrophosphate, adenylyl imidodiphosphate and GTP, which are inhibitors (competitive with TPP) of the overall pyruvate dehydrogenase reaction, did not appreciably affect TPP binding. 2. Initial-velocity patterns of the overall pyruvate dehydrogenase reaction obtained with varying TPP, CoA and NAD+ concentrations at a fixed pyruvate concentration were consistent with a sequential three-site Ping Pong mechanism; in the presence of oxaloacetate and citrate synthase to remove acetyl-CoA (an inhibitor of the overall reaction) the values of Km for NAD+ and CoA were 53+/- 5 muM and 1.9+/-0.2 muM respectively. Initial-velocity patterns observed with varying TPP concentrations at various fixed concentrations of pyruvate were indicative of either a compulsory order of addition of substrates to form a ternary complex (pyruvate-Enz-TPP) or a random-sequence mechanism in which interconversion of ternary intermediates is rate-limiting; values of Km for pyruvate and TPP were 25+/-4 muM and 50+/-10 nM respectively. The Kia-TPP (the dissociation constant for Enz-TPP complex calculated from kinetic plots) was close to the value of Kd-TPP (determined by direct binding studies). 3. Inhibition of the overall pyruvate dehydrogenase reaction by pyrophosphate was mixed non-competitive versus pyruvate and competitive versus TPP; however, pyrophosphate did not alter the calculated value for Kia-TPP, consistent with the lack of effect of pyrophosphate on the Kd for TPP. 4. Pyruvate dehydrogenase catalysed a TPP-dependent production of 14CO2 from [1-14C]pyruvate in the absence of NAD+ and CoA at approximately 0.35% of the overall reaction rate; this was substantially inhibited by phosphorylation of the enzyme both in the presence and absence of acetaldehyde (which stimulates the rate of 14CO2 production two- or three-fold). 5. Pyruvate dehydrogenase catalysed a partial back-reaction in the presence of TPP, acetyl-CoA and NADH. The Km for TPP was 4.1+/-0.5 muM. The partial back-reaction was stimulated by acetaldehyde, inhibited by pyrophosphate and abolished by phosphorylation. 6. Formation of enzyme-bound [14C]acetylhydrolipoate from [3-14C]pyruvate but not from [1-14C]acetyl-CoA was inhibited by phosphorylation. Phosphorylation also substantially inhibited the transfer of [14C]acetyl groups from enzyme-bound [14C]acetylhydrolipoate to TPP in the presence of NADH. 7...     

The effect of conjugated linoleic acid on arachidonic acid metabolism and eicosanoid production in human saphenous vein endothelial cells

The effects of a conjugated linoleic acid (CLA) mixture of single isomers (50:50, w/w, cis9,trans11:trans10,cis12) and the individual isomers on (a) the production of resting and calcium ionophore stimulated (14)C-eicosanoids and (b) the incorporation of (14)C-arachidonic acid (AA) into membrane phospholipids of human saphenous vein endothelial cells were investigated. The CLA mixture and the individual isomers were found to inhibit resting production of (14)C-prostaglandin F(2a) by 50, 43 and 40%, respectively. A dose dependent inhibition of stimulated (14)C-prostaglandins was observed with the CLA mixture (IC(50) 100 microM). The cis9,trans11 and trans10,cis12 (50 microM) isomers individually inhibited the overall production of stimulated (14)C-prostaglandins (between 35 and 55% and 23 and 42%, respectively). When tested at a high concentration (100 microM), cis9,trans11 was found to inhibit eicosanoid production in contrast to trans10,cis12 that caused stimulation. The overall degree of (14)C-AA incorporation into membrane phospholipids of the CLA (mixture and individual isomers) treated cells was found to be lower than that of control cells and the cis9,trans11 isomer was found to increase the incorporation of (14)C-AA into phosphatidylcholine. Docosahexaenoic acid, eicosapentaenoic acid and linoleic acid did not alter the overall degree of incorporation of (14)C-AA. The results of this study suggest that both isomers inhibit eicosanoid production, and although trans10,cis12 exhibits pro-inflammatory activity at high concentrations, the CLA mixture maintains its beneficial anti-inflammatory action that contributes to its anti-carcinogenic and anti-atherogenic properties.     

Involvement of alpha-cysteine-62 and beta-tryptophan-135 in human pyruvate dehydrogenase catalysis.

Pyruvate dehydrogenase (E1), a heterotetramer (alpha(2)beta(2)), is the first catalytic component of the mammalian pyruvate dehydrogenase complex (PDC). To investigate the roles of cysteine-62 of E1alpha (alphaC62) and tryptophan-135 of E1beta (betaW135) (identified previously as active site residues using chemical modifications) in E1 catalysis, two recombinant human E1 mutants were generated using site-directed mutagenesis: alphaC62A and betaW135L. Compared to wild-type, k(cat) values for alphaC62A and betaW135L measured by PDC assay were markedly reduced to 7.2 and 11. 6%, respectively. Apparent K(m) values for thiamin pyrophosphate (TPP) were increased approximately sixfold for both mutants, resulting in catalytic efficiency for TPP of only 1-2% of the wild-type E1. K(m) values for pyruvate increased only moderately (twofold). The alphaC62A and betaW135L mutants were less thermostable than wild-type E1. The conformations of the mutant apo-E1s determined by spectral analysis were different from that of the wild-type apo-E1. CD spectral analysis indicated that TPP binding was affected for both the alphaC62A and betaW135L mutant E1s. The substrate analogs, fluoropyruvate and bromopyruvate, were shown to be active site-directed inhibitors of human E1; in the absence of TPP, bromopyruvate (but not fluoropyruvate) inhibited human E1 due to SH-group modification. Pyruvate induced inactivation of human E1 could be restored by thiol reagents. Cysteine-62 (and maybe another group) is proposed to be involved in E1 inhibition by the substrate and substrate analogs. Taken together these results indicate that alphaC62 and betaW135 facilitate coenzyme binding, and alphaC62 could be near the substrate-binding site.     

Bromopyruvate as an active-site-directed inhibitor of the pyruvate dehydrogenase multienzyme complex from Escherichia coli

Bromopyruvate behaves as an active-site-directed inhibitor of the pyruvate decarboxylase (E1) component of the pyruvate dehydrogenase complex of Escherichia coli. It requires the cofactor thiamin pyrophosphate (TPP) and acts initially as an inhibitor competitive with pyruvate (Ki ca. 90 microM) but then proceeds to react irreversibly with the enzyme, probably with the thiol group of a cysteine residue. E1 catalyzes the decomposition of bromopyruvate, the enzyme becoming inactivated once every 40-60 turnovers. Bromopyruvate also inactivates the intact pyruvate dehydrogenase complex in a TPP-dependent process, but the inhibition is more rapid and is mechanistically different. Under these conditions, bromopyruvate is decarboxylated, and the lipoic acid residues in the lipoate acetyltransferase (E2) component become reductively bromoacetylated. Further bromopyruvate then reacts with the new thiol groups thus generated in the lipoic acid residues, inactivating the complex. If reaction with the lipoic acid residues is prevented by prior treatment of the complex with N-ethylmaleimide in the presence of pyruvate, the mode of inhibition reverts to irreversible reaction with the E1 component. In both types of inhibition of E1, reaction of 1 mol of bromopyruvate/mol of E1 chain is required for complete inactivation, and all the evidence is consistent with reaction taking place at or near the pyruvate binding site.     

A single amino acid residue contributes to distinct mechanisms of inhibition of the human multidrug transporter by stereoisomers of the dopamine receptor antagonist flupentixol.

Both cis and trans isomers of the dopamine receptor antagonist flupentixol inhibit drug transport and reverse drug resistance mediated by the human multidrug transporter P-glycoprotein (Pgp) with a stereoselective potency. The rate of ATP hydrolysis by Pgp and photoaffinity labeling of Pgp with the substrate analogue [125I]iodoarylazidoprazosin ([125I]IAAP) are modulated by each isomer in an opposite manner, suggesting different mechanisms for the inhibitory effect on drug transport. In this study we demonstrate that substitution of a single phenylalanine residue at position 983 (F983) with alanine (F983A) in putative transmembrane (TM) region 12 selectively affects inhibition of Pgp-mediated drug transport by both isomers of flupentixol. In F983A the stimulatory effect of cis(Z)-flupentixol and the inhibitory effect of trans(E)-flupentixol on ATP hydrolysis and [125I]IAAP labeling were significantly altered. This indicates that F983 contributes to inhibition of drug transport by both isomers of flupentixol and plays an important role in stimulation and inhibition of ATP hydrolysis and [125I]IAAP labeling by cis(Z)- and trans(E)-flupentixol, respectively. The near-wild-type level of drug transport by the F983A Pgp mutant dissociates susceptibility to inhibition by flupentixol from drug translocation, indicating the allosteric nature of the flupentixol interaction. The inhibitory effects of cyclosporin A on drug transport, drug-stimulated ATP hydrolysis, and [125I]IAAP labeling as well as the stimulatory effect of verapamil on ATP hydrolysis by Pgp were minimally affected by substitution of F983, suggesting no global alteration in the structural and functional integrity of the mutant. Taken together, our data suggest that distinct mechanisms of inhibition of Pgp-mediated drug transport by the cis and trans isomers of flupentixol are mediated through a common site of interaction.     

Utilizing temperature-sensitive association of Pluronic F-127 with lipid bilayers to control liposome-cell adhesion

Genetic defects in pyruvate dehydrogenase complex (PDC) cause lactic acidosis, neurological deficits, and often early death. Most mutations of PDC are localized in the alpha subunit of the pyruvate dehydrogenase (E1) component. We have kinetically characterized a patient's missense mutation alphaH44R in E1alpha by creating and purifying three recombinant human E1s (alphaH44R, alphaH44Q, and alphaH44A). Substitutions at histidine-15 resulted in decreased V(max) values (6% alphaH44R; 30% alphaH44Q; 90% alphaH44A) while increasing K(m) values for thiamine pyrophosphate (TPP) compared to wild-type (alphaH44R, 3-fold; alphaH44Q, 7-fold; alphaH44A, 10-fold). This suggests that the volume of the residue at site 15 is important for TPP binding and substitution by a residue with a longer side chain disrupts the active site more than the TPP binding site. The rates of phosphorylation and dephosphorylation of alphaH44R E1 by E1-kinase and phospho-E1 phosphatase, respectively, were similar to that of the wild-type E1 protein. These results provide a biochemical basis for altered E1 function in the alphaH44R E1 patient.     

Mutants that alter the covalent structure of catalase hydroperoxidase II from Escherichia coli.

The three-dimensional structures of two HPII variants, V169C and H392Q, have been determined at resolutions of 1.8 and 2.1 A, respectively. The V169C variant contains a new type of covalent bond between the sulfur atom of Cys(169) and a carbon atom on the imidazole ring of the essential His(128). This variant enzyme has only residual catalytic activity and contains heme b. The chain of water molecules visible in the main channel may reflect the organization of the hydrogen peroxide substrates in the active enzyme. Two alternative mechanisms, involving either compound I or free radical intermediates, are presented to explain the formation of the Cys-His covalent bond. The H392Q and H392E variants exhibit 75 and 25% of native catalytic activity, respectively. The Gln(392) variant contains only heme b, whereas the Glu(392) variant contains a mixture of heme b and cis and trans isomers of heme d, suggesting of a role for this residue in heme conversion. Replacement of either Gln(419) and Ser(414), both of which interact with the heme, affected the cis:trans ratio of spirolactone heme d. Implications for the heme oxidation mechanism and the His-Tyr bond formation in HPII are considered.     

Zinc(II) and cadmium(II) metal complexes of thiamine pyrophosphate and 2-(α-hydroxyethyl)thiamine pyrophosphate: models for activation of pyruvate decarboxylase

Metal complexes of thiamine pyrophosphate (TPP) of the general formula [M₂(TPPH)₂Cl₂].4H₂O (M =Zn²⁺, Cd²⁺) were isolated from methanolic solutions and characterized by elemental analysis, FT-IR, and multinuclear NMR spectroscopies. The data provide evidence for the bonding of the metals to the N(1') atom of the pyrimidine ring and to the pyrophosphate group. The stability constant measurements of TPP and 2-(α-hydroxyethyl)thiamine pyrophosphate (HETPP) metal complexes in aqueous solution imply the formation of dimeric complex species similar to the isolated solid products. They indicate also that HETPP forms more stable metal complexes than does TPP. To evaluate the coenzyme action of TPP and HETPP metal complexes, enzymic studies have been done using pyruvate decarboxylase apoenzyme. TPP metal complexes do not bind to the apoenzyme, unlike the Zn(II)-HETPP complex which can act as coenzyme. Considering these results, possible functional implications for thiamine involvement in catalysis are discussed. Received 13 September 1999 / Accepted 4 January 2000     

Pyruvate decarboxylase is like acetolactate synthase (ILV2) and not like the pyruvate dehydrogenase E1 subunit

Protein sequences of pyruvate decarboxylase (PDC) derived from cloned yeast (Saccharomyces cerevisiae) and bacterial (Zymomonas mobilis) genes were compared with each other and with sequence databases. Extensive sequence similarities were found between them and with two others: cytochrome-linked pyruvate oxidase from Escherichia coli and acetolactate synthase (ilvI in E. coli; ILV2 gene in S. cerevisiae). All catalyse decarboxylation of pyruvate using thiamine pyrophosphate (TPP) as cofactor. General overall similarity suggests common ancestry for these enzymes. None of the sequences was similar to the E1 component of pyruvate dehydrogenase from E. coli which also decarboxylates pyruvate with the help of TPP.     

Probing the mechanism of inactivation of human pyruvate dehydrogenase by phosphorylation of three sites

Activity of the mammalian pyruvate dehydrogenase complex (PDC) is regulated by phosphorylation-dephosphorylation of three serine residues (designated site 1, Ser-264; site 2, Ser-271; site 3, Ser-203) in the alpha subunit of the pyruvate dehydrogenase (E1) component. Substitutions of the phosphorylation sites were generated by site-directed mutagenesis. Glutamate (S1E) and aspartate (S1D) substitutions at site 1 resulted in the complete loss of PDC activity; however, these mutants were variably active in the decarboxylation and 2,6-dichlorophenolindophenol assays. S1Q had only 3% of wild-type PDC activity. The apparent K(m) values for pyruvate increased for the mutants of site 1 when determined in the 2,6-dichlorophenolindophenol assay. The substitutions at sites 2 and 3 caused only moderate reductions in activity in the three assays. S3E had a 27-fold increase in the apparent K(m) for thiamine pyrophosphate and 8-fold increase in the K(i) for pyrophosphate. Site 3 was almost completely protected from phosphorylation by thiamine pyrophosphate. The results show that the size rather than negative charge of the substituted amino acid residue affects the active site of E1 and that modification of each of the three serine residues affect the active site in a site-specific manner for its ability to bind the cofactor and substrates.     

Crystal structure of 1-deoxy-D-xylulose 5-phosphate synthase, a crucial enzyme for isoprenoids biosynthesis

Isopentenyl pyrophosphate (IPP) is a common precursor for the synthesis of all isoprenoids, which have important functions in living organisms. IPP is produced by the mevalonate pathway in archaea, fungi, and animals. In contrast, IPP is synthesized by a mevalonate-independent pathway in most bacteria, algae, and plant plastids. 1-Deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the first and the rate-limiting step of the mevalonate-independent pathway and is an attractive target for the development of novel antibiotics, antimalarials, and herbicides. We report here the first structural information on DXS, from Escherichia coli and Deinococcus radiodurans, in complex with the coenzyme thiamine pyrophosphate (TPP). The structure contains three domains (I, II, and III), each of which bears homology to the equivalent domains in transketolase and the E1 subunit of pyruvate dehydrogenase. However, DXS has a novel arrangement of these domains as compared with the other enzymes, such that the active site of DXS is located at the interface of domains I and II in the same monomer, whereas that of transketolase is located at the interface of the dimer. The coenzyme TPP is mostly buried in the complex, but the C-2 atom of its thiazolium ring is exposed to a pocket that is the substrate-binding site. The structures identify residues that may have important roles in catalysis, which have been confirmed by our mutagenesis studies.     

Differential effects of two mutations at arginine-234 in the alpha subunit of human pyruvate dehydrogenase.

The most common mutation in the alpha subunit of the pyruvate dehydrogenase (E1) component of the human pyruvate dehydrogenase complex (PDC) is arginine-234 to glycine and glutamine in 12 and 3 patients, respectively. Interestingly, these two mutations at the same amino acid position cause E1 (and hence PDC) deficiency by apparently different mechanisms. Recombinant human R234Q E1 had similar V(max) (25.7 +/- 4.4 units/mg E1) and apparent K(m) (101 +/- 4 nM) values for TPP as recombinant wild-type human E1, while R234G E1 had no significant change in V(max) (33.6 +/- 4.7 units/mg E1) but had a 7-fold increase in its apparent K(m) value for TPP (497 +/- 25 nM). Both of the R234 mutant proteins had similar apparent K(m) values for pyruvate. Both R234Q and R234G mutant proteins displayed similar phosphorylation rates of sites 1 and 2 by pyruvate dehydrogenase kinase 2 (PDK2) and site 3 by PDK1 compared to wild-type E1. Phosphorylated R234Q E1, R234G E1, and wild-type E1 also had similar dephosphorylation rates of sites 1 and 2 by phosphopyruvate dehydrogenase phosphatase 1. The rate of dephosphorylation of site 3 was about 50% for R234Q E1 and without a significant change for R234G E1 compared to the wild type. The data indicate that the patients with the R234G E1 mutation are symptomatic due to a decreased ability of this mutant protein to bind TPP, whereas the patients with the R234Q E1 mutation are symptomatic due to a decreased rate of dephosphorylation of site 3, hence keeping the enzyme in a phosphorylated/inactivated form.     

A selective 19F nuclear magnetic resonance spectroscopic method for the assay of the neuroleptic drug cis(Z)-flupentixol in human serum

This investigation was carried out to evaluate 19F nuclear magnetic resonance as an analytical tool for the measurement of the cis(Z) and trans(E) stereoisomers of the antipsychotic drug flupentixol in human serum. The method is based on the integration of appropriate signals of both analytes and an internal standard. The proposed method was applied to the analysis of real samples without any interference, manipulation of large samples, and lengthy instrument time. Experimental parameters were selected to optimize accuracy, precision, and analysis time. The calibration curves in human serum matrix were linear for cis(Z)- and trans(E)-flupentixol over the ranges 4.0-50.0 and 2.6-25.0 microg/mL, respectively, with respective minimum detectable limits (S/N=3) of 1.67 and 1.72 microg/mL. The method was validated through spike and recovery for the two isomers of flupentixol from a human serum matrix.     

Characterization of point mutations in patients with pyruvate dehydrogenase deficiency: role of methionine-181, proline-188, and arginine-349 in the alpha subunit.

Human pyruvate dehydrogenase (E1), a heterotetramer (alpha2beta2), is the first component of the pyruvate dehydrogenase complex (PDC). E1 catalyzes the thiamin pyrophosphate (TPP)-dependent decarboxylation of pyruvate and the reductive acetylation of the dihydrolipoamide acetyltransferase component. Site-directed mutagenesis was employed to recreate three point mutations in the alpha subunit identified in E1-deficient patients, M181V, R349H, and P188L (P188A mutant E1 was used because of the very low level of expression of P188L), to investigate the functional roles of these three amino acid residues. P188A mutant E1 was much less thermostable than the wild-type E1. The kcats of M181V and P188A mutant E1s determined in the PDC reaction were 38 and 24% of that of the wild-type enzyme, respectively. The apparent Km for TPP for M181V increased significantly (approx 250-fold when determined in the PDC assay), while the apparent Km for pyruvate increased by only about 3-fold. In contrast, P188A had similar Kms for the coenzyme and the substrate as the wild-type. Km values for R349H were not determined due to the extremely low activity of this mutant (1.2% of the wild-type E1-specific activity measured in the PDC assay). Wild-type E1 displayed a lag phase in the progress curve of the PDC reaction measured in the presence of low TPP concentrations (below 1 microM) only. All mutants had a lag phase that was not eliminated even at very high TPP concentrations, suggesting modifications in the conformation of the active site. Kinetic analysis indicated thiamin 2-thiothiazolone pyrophosphate (ThTTPP) to be an intermediate analog for wild-type human E1. M181V required a higher concentration of ThTTPP for inactivation than the wild-type and P188A E1s. The results of circular dichroism spectropolarimetry in the far UV region indicated that there were no major changes in the secondary structure of M181V, P188A, and R349H E1s. These mutant enzymes exhibited negative dichroic spectra at about 330 nm only in the presence of high TPP concentrations. This study suggests that arginine-349 is critical for E1's activity, methionine-181 is involved in the binding of TPP, and proline-188 is necessary for structural integrity of E1.     

Closed site complexes of adenine phosphoribosyltransferase from Giardia lamblia reveal a mechanism of ribosyl migration

The adenine phosphoribosyltransferase (APRTase) from Giardia lamblia was co-crystallized with 9-deazaadenine and sulfate or with 9-deazaadenine and Mg-phosphoribosylpyrophosphate. The complexes were solved and refined to 1.85 and 1.95 A resolution. Giardia APRTase is a symmetric homodimer with the monomers built around Rossman fold cores, an element common to all known purine phosphoribosyltransferases. The catalytic sites are capped with a small hood domain that is unique to the APRTases. These structures reveal several features relevant to the catalytic function of APRTase: 1) a non-proline cis peptide bond (Glu(61)-Ser(62)) is required to form the pyrophosphate binding site in the APRTase.9dA.MgPRPP complex but is a trans peptide bond in the absence of pyrophosphate group, as observed in the APRTase.9dA.SO4 complex; 2) a catalytic site loop is closed and fully ordered in both complexes, with Glu(100) from the catalytic loop acting as the acid/base for protonation/deprotonation of N-7 of the adenine ring; 3) the pyrophosphoryl charge is neutralized by a single Mg2+ ion and Arg(63), in contrast to the hypoxanthine-guanine phosphoribosyltransferases, which use two Mg2+ ions; and 4) the nearest structural neighbors to APRTases are the orotate phosphoribosyltransferases, suggesting different paths of evolution for adenine relative to other purine PRTases. An overlap comparison of AMP and 9-deazaadenine plus Mg-PRPP at the catalytic sites of APRTases indicated that reaction coordinate motion involves a 2.1-A excursion of the ribosyl anomeric carbon, whereas the adenine ring and the 5-phosphoryl group remained fixed. G. lamblia APRTase therefore provides another example of nucleophilic displacement by electrophile migration.     

Effects of pyruvate dehydrogenase subunits overexpression on the α-ketoglutarate production in Yarrowia lipolytica WSH-Z06

Yarrowia lipolytica WSH-Z06 harbours a promising capability to oversynthesize α-ketoglutarate (α-KG). Its wide utilization is hampered by the formation of high concentrations of pyruvate. In this study, a metabolic strategy for the overexpression of the α and β subunits of pyruvate dehydrogenase E1, E2 and E3 components was designed to reduce the accumulation of pyruvate. Elevated expression level of α subunit of E1 component improved the α-KG production and reduced the pyruvate accumulation. Due to a reduction in the acetyl-CoA supply, neither the growth of cells nor the synthesis of α-KG was restrained by the overexpression of β subunit of E1, E2 and E3 components. Furthermore, via the overexpression of these thiamine pyrophosphate (TPP)-binding subunits, the dependency of pyruvate dehydrogenase on thiamine was diminished in strains T1 and T2, in which α and β subunits of E1 component were separately overexpressed. In these two recombinant strains, the accumulation of pyruvate was insensitive to variations in exogenous thiamine. The results suggest that α-KG production can be enhanced by altering the dependence on TPP of pyruvate dehydrogenase and that the competition for the cofactor can be switched to ketoglutarate dehydrogenase via separate overexpression of the TPP-binding subunits of pyruvate dehydrogenase. The results presented here provided new clue to improve α-KG production.     

Regulatory effect of thiamin pyrophosphate on pig heart pyruvate dehydrogenase complex

The kinetic behavior of pig heart pyruvate dehydrogenase complex (PDC) containing bound endogenous thiamin pyrophosphate (TPP) was affected by exogenous TPP. In the absence of exogenous TPP, a lag phase of the PDC reaction was observed. TPP added to the PDC reaction medium containing Mg2+ led to a disappearance of the lag phase, inducing strong reduction of the Km value for pyruvate (from 76.7 to 19.0 microM) but a more moderate decrease of Km for CoA (from 12.2 to 4.3 microM) and Km for NAD+ (from 70.2 to 33.6 microM), with no considerable change in the maximum reaction rate. Likewise, thiamin monophosphate (TMP) decreased the Km value of PDC for pyruvate, but to a lesser extent (from 76.7 to 57.9 microM) than TPP. At the unsaturating level of pyruvate, the A50 values for TPP and TMP were 0.2 microM and 0.3 mM, respectively. This could mean that the effect of TPP on PDC was more specific. In addition, exogenous TPP changed the UV spectrum and lowered the fluorescence emission of the PDC containing bound endogenous TPP in its active sites. The data obtained suggest that TPP plays, in addition to its catalytic function, the important role of positive regulatory effector of pig heart PDC.     

"Plasmalogen-type" cyclic acetals: formation and conformation of the 1,3-dioxanes and 1,3-dioxolanes from 1-0-cis-alk-1'-enyl-sn-glycerols

Acid-catalyzed cyclization of 1-O-cis-alk-1'-enyl-sn-glycerol produced four structurally and geometrically isomeric long-chain cyclic acetals of glycerol. The isomers were isolated by adsorption and gas-liquid chromatography and were identified as cis-2-alkyl-5-hydroxy-1,3-dioxane (Ia), trans-2-alkyl-5-hydroxy-1,3-dioxane (IIa), cis-2-alkyl-4-hydroxymethyl-1,3-dioxolane (IIIa), and trans-2-alkyl-4-hydroxymethyl-1,3-dioxolane (IVa). The structure of each isomer was established by chemical and spectroscopic methods. Cyclization with p-toluenesulfonic acid in boiling benzene led to a thermodynamically equilibrated mixture of isomers Ia-IVa in which the cis isomers predominated. Cyclization in acetic acid was found to be kinetically controlled, and formation of the trans isomers was relatively favored. Rearrangement of the cyclic acetal isomers did not occur in acetic acid; hence, optically active five-membered ring acetals were prepared.     

Thiamine-responsive pyruvate dehydrogenase deficiency in two patients caused by a point mutation (F205L and L216F) within the thiamine pyrophosphate binding region

The human pyruvate dehydrogenase complex (PDHC) catalyzes the thiamine-dependent decarboxylation of pyruvate. Thiamine treatment is very effective for some patients with PDHC deficiency. Among these patients, five mutations of the pyruvate dehydrogenase (E1)alpha subunit have been reported previously: H44R, R88S, G89S, R263G, and V389fs. All five mutations are in a region outside the thiamine pyrophosphate (TPP)-binding region of the E1alpha subunit.We report the biochemical and molecular analysis of two patients with clinically thiamine-responsive lactic acidemia. The PDHC activity was assayed using two different concentrations of TPP. These two patients displayed very low PDHC activity in the presence of a low (1 x 10(-4) mM) TPP concentration, but their PDHC activity significantly increased at a high (0.4 mM) TPP concentration. Therefore, the PDHC deficiency in these two patients was due to a decreased affinity of PDHC for TPP. Treatment of both patients with thiamine resulted in a reduction in the serum lactate concentration and clinical improvement, suggesting that these two patients have a thiamine-responsive PDHC deficiency. The DNA sequence of these two male patients' X-linked E1alpha subunit revealed a point mutation (F205L and L216F) within the TPP-binding region in exon 7.