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[Coenzyme-binding sites of the brain pyruvate dehydrogenase complex].

It is shown that the relative amount of the holoenzyme in the highly purified pyruvate dehydrogenase complex from the bovine brain is higher when the enzyme activity is assayed in the reaction of nonoxidative formation of acetaldehyde as compared to the pyruvate: NAD+ reductase reaction. The S0.5 values for thiamine pyrophosphate are as following: (TPP) (0.314 +/- 0.22) x 10(-7) M with reaction of nonoxidative formation of acetaldehyde, (0.188 +/- 0.08) x 10(-6) M and (1.65 +/- 1.16) x 10(-6) M in case of the pyruvate: NAD+ reductase reaction. TPP in the concentration of (0.5-6.0) x 10(-7) M completely protects the sites of nonoxidative formation of acetaldehyde from modification by the coenzyme analogs, 4'-oxythiamine pyrophosphate and tetrahydrothiamine pyrophosphate. However, the pyruvate: NAD+ reductase activity of the pyruvate dehydrogenase complex is inhibited in this case by 30-34%. The data obtained suggest that in contrast to the pyruvate: NAD+ reductase reaction the conversion of pyruvate to acetaldehyde occurs by the sites which tightly bound TPP.

Animals↗

PubMed1975

Acetoin degradation in Bacillus subtilis by direct oxidative cleavage.

Acetate and acetaldehyde can be detected as products of the oxidative dissimilation of acetoin in Bacillus subtilis extracts. They arise as the result of the direct cleavage of acetoin without a previous oxidation to diacetyl. This can be deduced from the following observations: (a) no diacetyl was detected in acetoin dissimilation experiments in vitro and (b) methylacetoin, an acetoin analogue which can not be oxidized to the diketone, also undergoes oxidative splitting, yielding acetone and acetate. The splitting reaction requires thiamine pyrophosphate as a cofactor, suggesting that the oxidative step occurs, as known for similar reactions, by the electron transfer from hydroxyethylthiamine pyrophosphate to a proper acceptor, which in vitro can be replaced by dichlorophenolindophenol. In vivo the final product of the oxidation of hydroxyethylthiamine pyrophosphate is activated acetate. A mutant which lacks acetoin-cleaving activity can not reutilize the acetoin accumulated after growth in glucose. This corroborates the actual importance of the cleavage reaction for acetoin dissimilation. The enzyme diacetylmethylcarbinol synthase, thought to be responsible for the formation of diacetylmethylcarbinol from diacetyl, probably is identical to the enzyme catalyzing the cleavage of acetoin.

Acetoin↗

PubMed1992

Isolation, characterization, and physiological role of the pyruvate dehydrogenase complex and alpha-acetolactate synthase of Lactococcus lactis subsp. lactis bv. diacetylactis.

The pyruvate dehydrogenase complex of Lactococcus lactis subsp. lactis bv. diacetylactis has a specific activity of 6.6 U/mg and a Km of 1 mM for pyruvate. The specific activities of E2 and E3 in the complex are 30 and 0.36 U/mg, respectively. The complex is very sensitive to NADH inhibition and consists of four subunits: E1 alpha (44 kDa), E1 beta (35 kDa), E2 (73 kDa), and E3 (60 kDa). The L. lactis alpha-acetolactate synthase has a specific activity of 103 U/mg and a Km of 50 mM for pyruvate. Thiamine pyrophosphate (Km = 3.2 microM) and divalent cations are essential for activity. The native enzyme measures 172 kDa and consists of 62-kDa monomers. The role of both enzymes in product formation is discussed in view of NADH inhibition and competition for pyruvate.

Acetolactate Synthase↗

PubMed1996

The thiJ locus and its relation to phosphorylation of hydroxymethylpyrimidine in Escherichia coli.

Extracts from Escherichia coli K-12 contained two distinct enzymes capable of catalysing the phosphorylation of hydroxymethylpyrimidine (HMP) to HMP monophosphate: pyridoxine kinase (EC 2.7.1.35) and an enzyme that has not previously been genetically analysed, HMP kinase (EC 2.7.1.49). Two distinct genes, pdxL and thiJ, specify the activities of the former and latter enzymes, respectively. The inactivation of both genes by independent mutations in the same cell resulted in the complete loss of HMP kinase activity. Experiments with a series of strains that carry mutations in thiC, thiC pdxB, thiC pdxB pdxL and thiC pdxB pdxL thiJ revealed that the ability of the double mutant (pdxL thiJ) to utilize HMP in thiamin pyrophosphate biosynthesis was restored by introducing the wild-type allele corresponding to the thiJ mutation. The thiJ locus was mapped on the chromosome near the thiD and thiM loci, which govern the activities of phosphomethylpyrimidine kinase (EC 2.7.4.7) and hydroxyethylthiazole kinase (EC 2.7.1.50), respectively.

Chromosome Mapping↗

PubMed1976

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...

Acetaldehyde↗

PubMed1990

The relationship between erythrocyte transketolase activity and the 'TPP effect' in Wernicke's encephalopathy and other thiamine deficiency states.

Patients (n = 104) were judged to be thiamine deficient by the criteria of erythrocyte transketolase activity (ETK) less than 0.6 U/g of hemoglobin, or greater than 17% increase in this activity on addition of thiamine pyrophosphate in vitro (TPP effect). ETK activated by TPP in vitro (AETK) was related to ETK by a linear regression of slope greater than or equal to 1, implying that transketolose apoenzyme (apoTK) was constant or decreased as ETK decreased. For most patient groups the value of apoTK was 0.1 U/g and the slope 1.033 to 1.050. In the subgroup of non-vomiting drinkers with Wernicke's encephalopathy (WE), the slope of the linear regression of AETK on ETK was 1.21, so that apoTK decreased as ETK decreased. Comparison of these data is consistent with a difference in the TK of WE drinkers from that of others. Generally, any variation of TPP effect was due only to variation of ETK. We recommend measurement of ETK, without TPP effect, for the assessment of thiamine nutrition.

Enzyme Activation↗

PubMed1986

Effect of liposomes sensitized with methotrexate-gamma-dimyristoylphosphatidylethanolamine on cells that are resistant to methotrexate.

This study compares the ability of methotrexate and liposomes, in which the drug is anchored to the lipid bilayers via methotrexate-gamma-dimyristoylphosphatidylethanolamine, to inhibit proliferation of human leukemic cells (CEM/O) and cells derived from this line that are resistant to methotrexate because of either a defective transport system (CEM/MTX cells) or elevated levels of dihydrofolate reductase (CEM/R1 cells). Whereas CEM/O and CEM/MTX cells show a 120-fold difference in their susceptibility to methotrexate (as measured by the incorporation of tritiated deoxyuridine into DNA), both lines are equally sensitive to the liposomes. In contrast, proliferation of CEM/MTX cells is not inhibited significantly by methotrexate-gamma-glycerophosphorylethanolamine (MTX-gamma-glyceroPE), the water-soluble analog of MTX-gamma-DMPE. Both the ability of the liposomes to circumvent the transport defect, and the inability of MTX-gamma-glyceroPE to do so, were anticipated on the basis of previous experiments which show that thiamine pyrophosphate could antagonize inhibition of mouse 3T3 and L1210 cell proliferation by methotrexate and MTX-gamma-glyceroPE, but not inhibition by liposomes. Human cells (CEM/O) behave similarly. The present experiments also suggest that liposomes prepared with MTX-gamma-DMPE can partially reverse the methotrexate resistance of CEM/R1 cells that is due to overproduction of the target enzyme.

Cell Division↗

PubMed1993

Structure of the thiamine- and flavin-dependent enzyme pyruvate oxidase.

Pyruvate oxidase from Lactobacillus plantarum is a tetrameric enzyme that decarboxylates pyruvate, producing hydrogen peroxide and the energy-storage metabolite acetylphosphate. Structure determination at 2.1 angstroms showed that the cofactors thiamine pyrophosphate (TPP) and flavin adenine dinucleotide (FAD) are bound at the carboxyl termini of six-stranded parallel beta sheets. The pyrophosphate moiety of TPP is bound to a metal ion and to a beta alpha alpha beta unit corresponding to an established sequence fingerprint. The spatial arrangement of TPP and FAD suggests that the oxidation of the oxyethyl intermediate does not occur by hydride displacement but rather by a two-step transfer of two electrons.

Amino Acid Sequence↗

PubMed1971

Relationship of nervous tissue transketolase to the neuropathy in chronic uremia.

Patients with chronic uremia develop neurologic defects which are similar to the demyelinating lesions seen in thiamine deficiency. The present study describes inhibitory effects of uremic material on nervous tissue transketolase, a thiamine-dependent enzyme of the pentose phosphate pathway which has been reported to have functional importance in the metabolism of myelinated nervous structures. Transketolase activity (TKA) of normal human brain and spinal cord was measured by the conversion of ribose-5-phosphate (R5P) to sedoheptulose-7-phosphate (S7P). TKA was significantly inhibited by plasma, cerebrospinal fluid and low molecular weight dialysate fractions obtained from patients with uremic neuropathy, but not by samples from normal subjects. The specific effect on transketolase by uremic material was established by showing suppressed formation of S7P from R5P also in the presence of excess cofactor thiamine pyrophosphate and of the other substrate xylulose-5-phosphate. Uremic plasma likewise inhibited a partially purified transketolase preparation from bakers' yeast.31 of 35 chronic uremic patients with inhibition values between 10 and 84% before or during the early phase of intermittent hemodialysis had evidence of neuropathy. Data of clinical grading of the neurologic deficits and values of motor nerve conduction velocity revealed a correlation between the extent of uremic neuropathy and the degree of nervous tissue transketolase inhibition. Hemodialysis markedly reduced the inhibitory effects of the patients' plasma and the data indicate that uremic patients who received effective long-term dialysis treatment show a parallel decline of transketolase inhibition and uremic neuropathy.The findings demonstrate that in patients with chronic renal failure, low molecular weight factors accumulate and inhibit nervous tissue transketolase. This biochemical defect-uncorrectable by thiamine but reversible by dialysis-may interfere with the metabolism of myelin-supporting cells, and/or of the axonal metabolism of medullated structures, and may thus contribute to the degeneration of myelinated nerves seen with uremic neuropathy.

Adolescent↗

PubMed1976

Evaluation of methods of coenzyme activation of erythrocyte enzymes for detection of deficiency of vitamins B1, B2, and B6.

We describe optimized, ultraviolet spectrophotometric procedures for determination of erythrocyte transketolase, glutathione reductase, and aspartate aminotransferase activity, and their activation by their respective coenzymes--thiamine pyrophosphate, flavin-adenine dinucleotide, and pyridoxal-5-phosphate--as tests for vitamin B1, B2, and B6 deficiency. With these procedures we have investigated healthy subjects on normal and vitamin-supplemented diets, and a series of (mainly) alcoholic hospital in-patients. The enzyme procedures described have good precision and can be readily carried out in the routine laboratory. Abnormal transketolase activation correlated well with clinical evidence of vitamin B1 deficiency.

Aspartate Aminotransferases↗

PubMed1989

Cytotoxicities of two disulfide-bond-linked conjugates of methotrexate with monoclonal anti-MM46 antibody.

In studies on (antitumor antibody)-drug conjugates as potential antitumor agents, the amide derivatives of methotrexate (MTX) with cysteine and with 2-mercaptoethylamine (cysteamine) (MTX-Cys and MTX-MEA, respectively) were linked via a disulfide bond with a monoclonal antibody (alpha MM46) to a mouse mammary tumor MM46 with attached 3-(2-pyridyldithio) propionyl groups to give conjugates of MTX with alpha MM46 (MTX-Cys-SS-alpha MM46 and MTX-MEA-SS-alpha MM46, respectively). These two conjugates are both linked by a disulfide bond and are very similar in structure, but MTX-MEA-SS-alpha MM46 showed only weak in vitro cytotoxicity against MM46 cells, whereas MTX-Cys-SS-alpha MM46 had strong cytotoxicity. The cytotoxicity of the latter was comparable to that of the conventional direct MTX-alpha MM46 conjugate prepared with an MTX-active ester. However, this conjugate had a greater selectivity than that of the direct conjugate, calculated as the IC50 (concentration of a conjugate by MTX equivalence required for suppression of the number of viable MM46 cells to 50% of that of the untreated control) for the corresponding nonspecific conjugate divided by the IC50 for the alpha MM46 conjugate. The inhibitory activities of MTX-Cys and MTX-MEA on dihydrofolate reductase were similar. The cytotoxicity of MTX-Cys-SS-alpha MM46 was not affected by thiamine pyrophosphate, an inhibitor of the active transport of MTX across the cell membrane, but was decreased significantly by ammonium chloride, a lysosomotropic amine. However, the cytotoxicity was decreased only to a small extent by leupeptin, an inhibitor of lysosomal cysteine proteases cathepsins B, H, and L. These results suggest that the cytotoxicity is mediated by lysosomes, and may involve lysosomal enzymes other than cathepsins B, H, and L.

Animals↗

PubMed1979

Is lactate involved in phenformin-induced insulin secretion?

Phenformin at high doses (10 mg/l, 50 mg/l, and 100 mg/l) increased the insulin and lactate output rates by the isolated perfused rat pancreas. Glucagon secretion was not modified. There was a statistically significant correlation between the increase in insulin and lactate output rates induced by phenformin. Intra-pancreatic L (+) lactate concentrations induced by phenformin were in the range of sodium L (+) lactate concentrations which experimentally stimulated insulin secretion by the same preparation. Thiamin pyrophosphate and sodium dichloroacetate, which promote the aerobic metabolism of pyruvate, opposed the phenoformin induced increase in lactate output by the isolated perfused rat pancreas and provoked as well a decrease in insulin release. These results suggest that the increase in insulin secretion following the administration of phenformin at high concentrations can be explained, to a large extent, by the increase in the production of lactate ions.

Animals↗

PubMed1983

Interaction of baker's yeast transketolase modified by 2,3-butanedione with anionic and nonanionic substrates.

Baker's yeast apotransketolase modified by 2,3-butanedione binds thiamine pyrophosphate, a coenzyme of transketolase, and forms a charge transfer complex which can be identified by the CD spectrum. The enzyme retains thereby its ability to bind anionic and nonanionic substrates. The affinity of the modified enzyme for donor substrates changes insignificantly, whereas Vmax decreases 5-10-fold. The results of the study show that, although the arginine residue of the active center is not involved in substrate binding, it is essential for catalysis.

Anions↗

PubMed1973

Comparison of acetohydroxy-acid synthetases from two extreme thermophilic bacteria.

The acetohydroxy-acid synthetases from two extreme bacterial thermophiles, Thermus aquaticus and Bacillus sp., have been studied. The two enzymes have different pH optima, 8 and 6, respectively, and both are feedback inhibited by valine. The inhibition is of interest because it is not expressed below 60 C, but only at higher temperatures which are optimal for catalytic activity. Valine inhibition in T. aquaticus was noncompetitive, whereas in Bacillus sp., it was competitive. Isoleucine (10(-3) M) also inhibited the two enzymes, whereas leucine (10(-3) M) did not. There was no concerted feedback when the amino acids were added in together. The sensitivity of the enzymes to valine could not be removed by HgCl(2). Both enzymes required Mg(2+) and thiamine pyrophosphate for optimal activity, whereas only the enzyme from T. aquaticus required flavine adenine dinucleotide in addition. None of these cofactors was essential for the feedback inhibition caused by valine. The enzymes from both bacteria could be repressed, but only in the presence of all three branched-chain amino acids indicating that, as in Escherichia coli and Salmonella typhimurium, the repression system is multivalent.

Bacillus↗

PubMed1979

Abnormal pyruvate and alpha-ketoglutarate dehydrogenase complexes in a patient with lactic acidemia.

Activities of pyruvate decarboxylase (PDC), alpha-ketoglutarate decarboxylase (KGDC) and both the pyruvate and alpha-ketoglutarate dehydrogenase complexes (PDH complex and KGDH complex) were measured, and kinetic properties of PDC were studied in fibroblasts derived from normal individuals and from a 2-yr-old girl with congenital lactic acidemia and severe retardation of growth and development. The activities of PDC, KGDC, PDH complex, and KGDH complex in the patient were 1.12 +/- 0.12, 2.33 +/- 0.42, 9.00 +/- 0.50, and 16.46 +/- 1.57 and in controls 3.10 +/- 0.16, 5.36 +/- 0.56, 24.13 +/- 1.61 and 44.95 +/- 3.72 nmole/mg protein/hr. The optimum pH (6.0) and Michaelis constants (Km) for pyruvate of PDC (1.0-1.6 X 10(-5) M) were similar in fibroblasts of the patient and controls. PDC activity was more sensitive to denaturation by heat in the fibroblasts of the patient than those from controls, while heat denaturation curves of KGDC were similar in the patient and control. Higher concentrations of thiamine pyrophosphate (TPP) were required to protect PDC from heat denaturation in the patient. TPP was more easily removed from PDC in the patient than in the control by washing the fibroblasts with alkaline buffer. These results suggest that the PDC enzyme of the patient is in an altered molecular form, to which TPP is loosely bound. This particular constellation of abnormalities has not previously been reported in patients with lactic acidemia.

Acidosis↗

PubMed1982

Purification and properties of a transketolase responsible for formaldehyde fixation in a methanol-utilizing yeast, candida boidinii (Kloeckera sp.) No. 2201.

Dihydroxyacetone synthase, present in methanol-grown Candida boidinii (Kloeckera sp.) No. 2201, catalyzes the transfer of the glycolaldehyde group from xylulose 5-phosphate to formaldehyde to form glyceraldehyde 3-phosphate and dihydroxyacetone. This enzyme was purified to electrophoretic homogeneity and found to be a new type of transketolase. The molecular weight of the enzyme was estimated to be 190,000 by gel filtration. The enzyme appeared to be composed of four identical subunits (Mr, 55,000). Thiamin pyrophosphate and Mg2+ were required for the activity. The optimum pH was found to be 7.0. With xylulose 5-phosphate as the ketol-donor, aliphatic aldehydes (C1-C7), glycolaldehyde and glyceraldehyde were better acceptors than ribose 5-phosphate. The kinetic data were consistent with a ping-pong bi-bi mechanism. The Km values obtained were as follows: xylulose 5-phosphate, 1.0 mM; formaldehyde, 0.43 mM; glyceraldehyde 3-phosphate, 0.42 mM; and dihydroxyacetone, 0.52 mM.

Candida↗

PubMed1980

Taurine catabolism. II. biochemical and genetic evidence for sulfoacetaldehyde sulfo-lyase involvement.

Cell-free extracts of Pseudomonas aeruginosa. TAU-5 catalyze the cleavage of chemically or enzymatically synthesized sulfoacetaldehyde to form acetate and sulfite. The activity is enhanced by the presence of thiamine pyrophosphate. The sulfo-lyase responsible for this reaction has been partially purified 9-fold in order to separate it from taurine: pyruvate aminotransferase and to demonstrate its role in taurine catabolism. The sulfo-lyase is induced in organisms grown on taurine but not on other compounds tested. The induction occurs co-ordinately with the induction of the aminotransferase. Mutagenesis of the organism yielded a strain which lacks the sulfo-lyase and in incapable of growing on taurine. A revertant of this strain regained all the prototrophic characterics.

Acetaldehyde↗

PubMed1997

In vitro kinetic studies of formation of antigenic advanced glycation end products (AGEs). Novel inhibition of post-Amadori glycation pathways.

Nonenzymatic protein glycation (Maillard reaction) leads to heterogeneous, toxic, and antigenic advanced glycation end products ("AGEs") and reactive precursors that have been implicated in the pathogenesis of diabetes, Alzheimer's disease, and normal aging. In vitro inhibition studies of AGE formation in the presence of high sugar concentrations are difficult to interpret, since AGE-forming intermediates may oxidatively arise from free sugar or from Schiff base condensation products with protein amino groups, rather than from just their classical Amadori rearrangement products. We recently succeeded in isolating an Amadori intermediate in the reaction of ribonuclease A (RNase) with ribose (Khalifah, R. G., Todd, P., Booth, A. A., Yang, S. X., Mott, J. D., and Hudson, B. G. (1996) Biochemistry 35, 4645-4654) for rapid studies of post-Amadori AGE formation in absence of free sugar or reversibly formed Schiff base precursors to Amadori products. This provides a new strategy for a better understanding of the mechanism of AGE inhibition by established inhibitors, such as aminoguanidine, and for searching for novel inhibitors specifically acting on post-Amadori pathways of AGE formation. Aminoguanidine shows little inhibition of post-Amadori AGE formation in RNase and bovine serum albumin, in contrast to its apparently effective inhibition of initial (although not late) stages of glycation in the presence of high concentrations of sugar. Of several derivatives of vitamins B1 and B6 recently studied for possible AGE inhibition in the presence of glucose (Booth, A. A., Khalifah, R. G., and Hudson, B. G. (1996) Biochem. Biophys. Res. Commun. 220, 113-119), pyridoxamine and, to a lesser extent, thiamine pyrophosphate proved to be novel and effective post-Amadori inhibitors that decrease the final levels of AGEs formed. Our mechanism-based approach to the study of AGE inhibition appears promising for the design and discovery of novel post-Amadori AGE inhibitors of therapeutic potential that may complement others, such as aminoguanidine, known to either prevent initial sugar attachment or to scavenge highly reactive dicarbonyl intermediates.

Animals↗