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PubMed1988

Pyruvate dehydrogenase activity in osmotically shocked rat brain mitochondria: stimulation by oxaloacetate.

Pyruvate dehydrogenase complex activity (PDHC) measured by CO2 release isotopic assay has generally been much lower than activity measured by the spectrophotometric arylamine acetyltransferase assay (ArAT). Decarboxylation of [1-14C]pyruvate was measured in osmotically shocked rat brain cortical mitochondria. Activity is dependent on the concentration of the substrate pyruvate. Activity of 74.6 units +/- 12.3 SD (n = 22) was observed at 4 mM pyruvate (1 unit = 1 nmol pyruvate decarboxylated/min/mg protein). Activity was dependent on added NAD, CoA, and thiamine pyrophosphate, implying increased mitochondrial permeability after osmotic shock. Freeze/thaw with sonication of the mitochondrial preparation reduced PDHC activity to 11.5 units +/- 3.0 SD (n = 4). Oxaloacetate produced a marked stimulation of activity. The optimal assay contained 3 mM oxaloacetate, and without oxaloacetate activity fell to 15.4 units +/- 9.9 SD (n = 8). These studies highlight the importance of optimal substrate concentrations in the CO2 release isotopic PDHC method. Higher PDHC activity is found with intact mitochondria and thus activity values should be interpreted in the light of the presence or absence of intact mitochondria in individual preparations.

Animals↗
PubMed1989

Phosphorylated thiamine derivatives and cortical activity in the baboon Papio papio: effect of intermittent light stimulation.

The effect of intermittent light stimulation (ILS) on the distribution of thiamine derivatives in three brain areas (occipital, motor, and premotor) was compared in photosensitive and nonphotosensitive baboons. ILS induces paroxysmal discharges in the motor and premotor areas of photosensitive animals only. In baboons submitted to ILS, thiamine triphosphate (TTP) decreases in both photosensitive and nonphotosensitive animals; thiamine monophosphate (TMP) increases in photosensitive animals, which present ILS-induced paroxysmal discharges, whereas it is unaffected in nonphotosensitive animals. The variations are the most significant in the occipital (visual) cortex. A consumption of TTP may result from electrical activity induced by light stimulation in the occipital area. No correlation between ILS-induced paroxysmal activity and a decrease in TTP contents was found. However, photosensitive animals are affected differently from nonphotosensitive animals, as their content of TMP in the cerebral cortex increases on stimulation. However, as long as the exact role of thiamine compounds in relation to membrane excitability in the nervous system remains unknown, it is impossible to conclude whether the differences observed in the metabolism of thiamine compounds are the cause or the consequence of the photosensitivity in the baboon Papio papio.

Animals↗
PubMed1993

Metabolism of thiamine triphosphate in rat brain: correlation with chloride permeability.

Our results show that a net synthesis of thiamine triphosphate (TTP) can be demonstrated in vitro using rat brain extracts. The total homogenate was preincubated with thiamine or its diphosphate derivative (TDP), centrifuged, and washed twice. With TDP (1 mM) as substrate, a 10-fold increase in TTP content was observed in this fraction (nuclear fraction, membrane vesicles). A smaller, but significant, increase was observed in the P2 fraction (mitochondrial/synaptosomal fraction). In view of the low TTP content of our fractions, it was carefully assessed that authentic TTP was being formed. Incorporation of radioactivity from [beta-32P]TDP and [gamma-32P]ATP in TTP suggests that these two compounds are its precursors. Furthermore, TTP synthesis was inhibited by ADP and relatively low concentrations of Zn2+. These results suggest that TTP synthesis is catalyzed by an ATP:TDP transphosphorylase rather than by the cytoplasmic adenylate kinase that may be present in the vesicles. After osmotic lysis of the vesicles at alkaline pH, TTP was recovered in protein-bound form. Concomitantly, a soluble thiamine triphosphatase, with alkaline pH optimum, was also released from the vesicles. No net synthesis could be obtained in the cytosolic fraction or in detergent-solubilized systems. Like TTP synthesis, chloride permeability of the vesicles was increased when the homogenate had been incubated with thiamine and particularly with TDP. Our results suggest a regulatory role of TTP on chloride permeability, but the target remains to be characterized.

Animals↗
PubMed1993

Brain alpha-ketoglutarate dehydrogenase complex activity in Alzheimer's disease.

We measured the activity of the alpha-ketoglutarate dehydrogenase complex (alpha-KGDHC), a rate-limiting Krebs cycle enzyme, in postmortem brain samples from 38 controls and 30 neuropathologically confirmed Alzheimer's disease (AD) cases, in both the presence and absence of thiamine pyrophosphate (TPP), the enzyme's cofactor. Statistically significant correlations between brain pH and lactate levels and alpha-KGDHC activity in the controls were observed, suggesting an influence of agonal status on the activity of alpha-KGDHC. As compared with the controls, mean alpha-KGDHC activity, with added TPP, was significantly (p < 0.005) reduced in AD brain in frontal (-56%), temporal (-60%), and parietal (-68%) cortices, with the reductions (-25 to -53%) in the occipital cortex, hippocampus, amygdala, and caudate failing to reach statistical significance. In the absence of exogenously administered TPP, mean alpha-KGDHC activity was reduced to a slightly greater extent in all seven AD brain areas (-39 to -83%), with the reductions now reaching statistical significance in the four cerebral cortical areas and hippocampus. A statistically significant negative correlation was observed between alpha-KGDHC activity and neurofibrillary tangle count in AD parietal cortex, the brain area exhibiting the most marked reduction in enzyme activity; this suggests that the enzyme activity reduction in AD brain may be related to the disease process and severity. In each brain area examined, TPP produced a greater stimulatory effect on alpha-KGDHC activity in the AD group (23-280% mean stimulation) as compared with the controls (-4 to +50%); this TPP effect could be explained by reduced endogenous TPP levels in AD brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1992

Blood and serum thiamin and thiamin phosphate esters concentrations in patients with alcohol dependence syndrome before and after thiamin treatment.

The blood and serum concentrations of free thiamin and its three phosphate esters were determined concomitantly by a new high-performance liquid chromatography (HPLC) method in 30 patients with alcohol dependence syndrome on admission to hospital and 24 hr after thiamin injection. We studied 24 men and 6 women; mean age, 50 years (range 21 to 69); mean ethanol consumption during the last 30 days, 164 +/- 119 g/day. A control group included 40 healthy volunteers (25 men, 15 women), of whom 10 were given the same thiamin injection as were the patients. Thiamin monophosphate was significantly reduced in the patients compared with controls before treatment (men 2.9 +/- 2.3 and 5.9 +/- 3.1 nmol/liter) and after (8.1 +/- 5.1 and 19.5 +/- 8.1 nmol/liter). On admission, free thiamin and thiamin diphosphate were similar in controls and in patients in whole blood (B) and serum (S) and increased similarly after treatment (mean B-thiamin diphosphate in male patients: 149 +/- 64 to 238 +/- 88 nmol/liter, in controls: 179 +/- 40 to 289 +/- 18 nmol/liter). However, seven patients had extremely high free thiamin values. The phosphorylation ratio was lower in patients than in controls (p less than 0.05), before and after treatment. Finally, the mean B-diphosphate was lower in patients not taking vitamins (116 +/- 48 nmol/liter and 172 +/- 57 nmol/liter) and in patients with polyneuropathy (118 +/- 54 nmol/liter and 173 +/- 52 nmol/liter), compared with the other patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1976

Enzymes of carbohydrate metabolism in four human species of Leishmania: a comparative survey.

The occurrence and levels of activity of various enzymes of carbohydrate catabolism in culture forms (promastigotes) of 4 human species of Leishmania (L. brasiliensis, L. donovani, L. mexicana, and L. tropica) were compared. These organisms possess enzymes of the Embden-Meyerhof pathway but lack lactate dehydrogenase. No evidence could be found for the production of lactic acid by growing cultures and lactic acid could not be detected either in cell-free preparations or after incubation of cell-free extracts with pyruvate and NADH under appropriate conditions. All 4 species possess alpha-glycerophosphate dehydrogenase and alpha-glycerophosphate phosphatase which together could regenerate NAD, thus compensating for the absence of lactate dehydrogenase. The oxidative and nonoxidative reactions of the hexose monophosphate pathway are present in all 4 species. Cell-free extracts have pyruvate dehydrogenase activity which allows the entry of pyruvate into and its subsequent oxidation through the tricarboxylic acid cycle. All enzymes of this cycle, including a thiamine pyrophosphate dependent alpha-ketoglutarate dehydrogenase, are present. Both NAD and NADP-linked malate dehydrogenase activities are present. The isocitrate dehydrogenase is NADP specific. There is an active glutamate dehydrogenase which could compete with alpha-ketoglutarate dehydrogenase for the common substrate (alpha-ketoglutarate). Replenishment of C4 acids is accomplished by heterotrophic CO2 fixation catalyzed by pyruvate carboxylase. All 4 species have high levels of NADH oxidase activity. Several enzymes thus far not found in any species of Leishmania have been demonstrated. These are: phosphoglucose isomerase, triose phosphate isomerase, fructose-1, 6-diphosphatase, 3-phosphoglycerate kinase, enolase, alpha-glycerophosphate dehydrogenase, alpha-glycerophosphate phosphatase, pyruvate dehydrogenase complex, citrate synthase, aconitase, alpha-ketoglutarate dehydrogenase, glutamate dehydrogenase, and NADH oxidase.

Animals↗
PubMed1998

Microbial desulfonation.

Organosulfonates are widespread compounds, be they natural products of low or high molecular weight, or xenobiotics. Many commonly found compounds are subject to desulfonation, even if it is not certain whether all the corresponding enzymes are widely expressed in nature. Sulfonates require transport systems to cross the cell membrane, but few physiological data and no biochemical data on this topic are available, though the sequences of some of the appropriate genes are known. Desulfonative enzymes in aerobic bacteria are generally regulated by induction, if the sulfonate is serving as a carbon and energy source, or by a global network for sulfur scavenging (sulfate-starvation-induced (SSI) stimulon) if the sulfonate is serving as a source of sulfur. It is unclear whether an SSI regulation is found in anaerobes. The anaerobic bacteria examined can express the degradative enzymes constitutively, if the sulfonate is being utilized as a carbon source, but enzyme induction has also been observed. At least three general mechanisms of desulfonation are recognisable or postulated in the aerobic catabolism of sulfonates: (1) activate the carbon neighboring the C-SO3- bond and release of sulfite assisted by a thiamine pyrophosphate cofactor; (2) destabilize the C-SO3- bond by addition of an oxygen atom to the same carbon, usually directly by oxygenation, and loss of the good leaving group, sulfite; (3) an unidentified, formally reductive reaction. Under SSIS control, different variants of mechanism (2) can be seen. Catabolism of sulfonates by anaerobes was discovered recently, and the degradation of taurine involves mechanism (1). When anaerobes assimilate sulfonate sulfur, there is one common, unknown mechanism to desulfonate the inert aromatic compounds and another to desulfonate inert aliphatic compounds; taurine seems to be desulfonated by mechanism (1).

Alkanesulfonates↗
PubMed2002

Expression of the xylulose 5-phosphate phosphoketolase gene, xpkA, from Lactobacillus pentosus MD363 is induced by sugars that are fermented via the phosphoketolase pathway and is repressed by glucose mediated by CcpA and the mannose phosphoenolpyruvate phosphotransferase system.

Purification of xylulose 5-phosphate phosphoketolase (XpkA), the central enzyme of the phosphoketolase pathway (PKP) in lactic acid bacteria, and cloning and sequence analysis of the encoding gene, xpkA, from Lactobacillus pentosus MD363 are described. xpkA encodes a 788-amino-acid protein with a calculated mass of 88,705 Da. Expression of xpkA in Escherichia coli led to an increase in XpkA activity, while an xpkA knockout mutant of L. pentosus lost XpkA activity and was not able to grow on energy sources that are fermented via the PKP, indicating that xpkA encodes an enzyme with phosphoketolase activity. A database search revealed that there are high levels of similarity between XpkA and a phosphoketolase from Bifidobacterium lactis and between XpkA and a (putative) protein present in a number of evolutionarily distantly related organisms (up to 54% identical residues). Expression of xpkA in L. pentosus was induced by sugars that are fermented via the PKP and was repressed by glucose mediated by carbon catabolite protein A (CcpA) and by the mannose phosphoenolpyruvate phosphotransferase system. Most of the residues involved in correct binding of the cofactor thiamine pyrophosphate (TPP) that are conserved in transketolase, pyruvate decarboxylase, and pyruvate oxidase were also conserved at a similar position in XpkA, implying that there is a similar TPP-binding fold in XpkA.

Aldehyde-Lyases↗
PubMed2005

Physiological characterization of the ARO10-dependent, broad-substrate-specificity 2-oxo acid decarboxylase activity of Saccharomyces cerevisiae.

Aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae CEN.PK113-7D were grown with different nitrogen sources. Cultures grown with phenylalanine, leucine, or methionine as a nitrogen source contained high levels of the corresponding fusel alcohols and organic acids, indicating activity of the Ehrlich pathway. Also, fusel alcohols derived from the other two amino acids were detected in the supernatant, suggesting the involvement of a common enzyme activity. Transcript level analysis revealed that among the five thiamine-pyrophospate-dependent decarboxylases (PDC1, PDC5, PDC6, ARO10, and THI3), only ARO10 was transcriptionally up-regulated when phenylalanine, leucine, or methionine was used as a nitrogen source compared to growth on ammonia, proline, and asparagine. Moreover, 2-oxo acid decarboxylase activity measured in cell extract from CEN.PK113-7D grown with phenylalanine, methionine, or leucine displayed similar broad-substrate 2-oxo acid decarboxylase activity. Constitutive expression of ARO10 in ethanol-limited chemostat cultures in a strain lacking the five thiamine-pyrophosphate-dependent decarboxylases, grown with ammonia as a nitrogen source, led to a measurable decarboxylase activity with phenylalanine-, leucine-, and methionine-derived 2-oxo acids. Moreover, even with ammonia as the nitrogen source, these cultures produced significant amounts of the corresponding fusel alcohols. Nonetheless, the constitutive expression of ARO10 in an isogenic wild-type strain grown in a glucose-limited chemostat with ammonia did not lead to any 2-oxo acid decarboxylase activity. Furthermore, even when ARO10 was constitutively expressed, growth with phenylalanine as the nitrogen source led to increased decarboxylase activities in cell extracts. The results reported here indicate the involvement of posttranscriptional regulation and/or a second protein in the ARO10-dependent, broad-substrate-specificity decarboxylase activity.

Carboxy-Lyases↗
PubMed1985

Pyruvate dehydrogenase activity in Streptococcus mutans.

Streptococcus mutans NCTC 10449 and Escherichia coli K-12 strain 37 were grown under aerobic and anaerobic conditions. In cell extracts of both strains, pyruvate dehydrogenase activity dependent on thiamine pyrophosphate, coenzyme A, and NAD was shown. The enzyme was induced by pyruvate in the growth medium, and there was higher activity in aerobically grown cells than in anaerobically grown cells. Acetyl phosphate was a potent inhibitor of the activity. This inhibition was partly overcome by inorganic phosphate.

Ferricyanides↗
PubMed2006

PurF-independent phosphoribosyl amine formation in yjgF mutants of Salmonella enterica utilizes the tryptophan biosynthetic enzyme complex anthranilate synthase-phosphoribosyltransferase.

In Salmonella enterica, the biosynthetic pathways for the generation of purines and the essential cofactor thiamine pyrophosphate branch after sharing five enzymatic steps. Phosphoribosyl amine (PRA) is the first intermediate in the common portion of the pathway and is generated from phosphoribosylpyrophosphate and glutamine by the PurF enzyme (phosphoribosylpyrophosphate amidotransferase). A null mutation in yjgF allows PurF-independent PRA formation by an unknown mechanism. The tryptophan biosynthetic enzyme complex anthranilate synthase-phosphoribosyltransferase, composed of the TrpD and TrpE proteins, was shown to be essential for PRA formation in strains lacking both yjgF and purF. The activity generating PRA in a yjgF mutant background has features that distinguish it from the TrpDE-mediated PRA formation shown previously for this enzyme in strains with an active copy of yjgF. The data presented here are consistent with a model in which the absence of YjgF uncovers a new catalytic activity of TrpDE.

Anthranilate Phosphoribosyltransferase↗
PubMed1970

Involvement of the protocatechuate pathway in the metabolism of mandelic acid by Aspergillus niger.

Cell-free extracts of Aspergillus niger UBC 814 grown in the presence of dl-mandelate oxidized both d(-)- and l(+)-mandelate via benzoylformate and benzaldehyde to benzoate. dl-p-Hydroxymandelate was oxidized, presumably through a parallel pathway, to p-hydroxybenzoate. A particulate d(-)-mandelate dehydrogenase and a supernatant fraction l(+)-mandelate dehydrogenase converted their respective substrates to benzoylformate. Both flavine adenine dinucleotide and flavine mononucleotide showed a stimulatory effect on the activity of the l(+)-mandelate dehydrogenase. Benzoylformate was decarboxylated to benzaldehyde by an enzyme requiring thiamine pyrophosphate for maximal activity. Two benzaldehyde dehydrogenases dependent on nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), respectively, for their activity dehydrogenated benzaldehyde to benzoate. In the presence of reduced NADP (NADPH), benzoate was oxidized via p-hydroxybenzoate and protocatechuate. Reduced NAD could not replace NADPH. Sensitive methods of assay for d(-)-mandelate dehydrogenase and benzoylformate decarboxylase are described. The fungal pathway is compared with these systems in bacteria.

Aspergillus↗
PubMed1978

Reduction of nicotinamide adenine dinucleotide by pyruvate:lipoate oxidoreductase in anaerobic, dark-grown Rhodospirillum rubrum mutant C.

Cell extracts from fermentatively grown Rhodospirillum rubrum reduced about 80 nmol of nicotinamide adenine dinucleotide (NAD) per mg of protein per min under anaerobic conditions with sodium pyruvate. The reaction was specific for pyruvate and NAD; NAD phosphate was not reduced. Results indicated that pyruvate-linked NAD reduction occurred via pyruvate:lipoate oxidoreductase. The reaction required catalytic amounts of both coenzyme A and thiamine pyrophosphate. Addition of sodium arsenite inhibited enzyme activity by 90%. Pyruvate:lipoate oxidoreductase was the only system detected in anaerobic, dark-grown R. rubrum cell extracts which operated to produce reduced NAD. The low activity of the enzyme system suggested that it was not quantitatively important in ATP formation.

Anaerobiosis↗
PubMed1981

Purification of a branched-chain keto acid dehydrogenase from Pseudomonas putida.

We purified branched-chain keto acid dehydrogenase to a specific activity of 10 mumol/min per mg of protein from Pseudomonas putida grown on valine. The purified enzyme was active with 2-ketoisovalerate, 2-ketoisocaproate, and 2-keto-3-methylvalerate in a ratio of 1.0:0.8:0.7 but showed no activity with either pyruvate or 2-ketoglutarate. There were four polypeptides in the purified enzyme (molecular weights, 49,000, 46,000, 39,000, and 37,000). The purified enzyme was deficient in the specific lipoamide dehydrogenase produced during growth on valine (molecular weight, 49,000). Branched-chain keto acid dehydrogenase required L-valine, oxidized nicotinamide adenine dinucleotide, coenzyme A, thiamine pyrophosphate, and magnesium chloride. A partially purified preparation catalyzed the oxidation of 2-keto-[1-14C]isovalerate to [14C]carbon dioxide, isobutyryl-coenzyme A, and reduced nicotinamide adenine dinucleotide in equimolar amounts. Both the Km and the Vmax for 2-ketoisovalerate were affected by the addition of L-valine to the assay mixture. However, only the Vmax values for oxidized nicotinamide adenine dinucleotide and coenzyme A were affected when L-valine was present. This suggested that valine acted by affecting the binding of branched-chain keto acids to subunit E1 of the complex.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1987

Sulfometuron methyl-sensitive and -resistant acetolactate synthases of the archaebacteria Methanococcus spp.

The herbicide sulfometuron methyl (SM) inhibited growth of some methanococci. Of 28 strains tested, the growth of 7 was completely inhibited by 0.55 mM SM. Growth of an additional 14 strains was partially inhibited, and the growth of 7 strains was unaffected by this concentration of SM. In some cases, the branched-chain amino acids protected growth. Growth inhibition was correlated with the Ki for SM of acetolactate synthase (ALS). For the enzymes from bacteria representative of the sensitive, partially resistant, and resistant methanococci (Methanococcus aeolicus, Methanococcus maripaludis, and Methanococcus voltae, respectively), the Ki for SM was 0.0012, 0.34, and greater than 1.0 mM, respectively. Inhibition was uncompetitive with respect to pyruvate. Based on these observations, ALS appeared to be the major if not the sole site of action of SM in the methanococci. The sensitivity of the ALS from these three methanococci to feedback inhibition by branched-chain amino acids was also quite different. Although all three were sensitive to feedback inhibition by valine, the Ki varied 20-fold, from 0.01 to 0.22 mM. Moreover, only the ALS from M. maripaludis was sensitive to inhibition by leucine, and the Ki was 1.8 mM. The Ki for isoleucine for the ALS from both M. maripaludis and M. voltae was about 0.1 mM. The ALS from M. aeolicus was not inhibited by isoleucine. In other respects, the ALSs from the methanococci were very similar. After dialysis, thiamine pyrophosphate but not FAD and Mg2+ was required for maximal activity, and they were all rapidly inactivated by oxygen. Although the methanococcal ALSs exhibited diverse properties, the range of catalytic and regulatory properties closely resembled those of the eubacterial enzymes.

Acetolactate Synthase↗
PubMed1988

Enhanced acetohydroxy acid synthase III activity in an ilvH mutant of Escherichia coli K-12.

The acetohydroxy acid synthase III isozyme, which catalyzes the first common step in the biosynthesis of isoleucine, leucine, and valine in Escherichia coli K-12, is composed of two subunits, the ilvI and ilvH gene products. A missense mutation in ilvH (ilvH612), which reduced the sensitivity of the enzyme to the end product inhibition by valine, also increased its specific activity and lowered the Km for alpha-acetolactate synthesis. The mutation increased the sensitivity of acetohydroxy acid synthase III to dialysis and heat treatment and reduced the requirement for thiamine pyrophosphate addition to the assay mixture for activity. A strain carrying the ilvH612 mutation grew better than a homologous ilvH+ strain in the presence of leucine. The data indicate that this is a consequence of a more active acetohydroxy acid synthase III isozyme rather than the result of an alteration of the leucine-mediated repression of the ilvIH operon.

Acetolactate Synthase↗
PubMed1989

Purification and characterization of oxalyl-coenzyme A decarboxylase from Oxalobacter formigenes.

Oxalyl-coenzyme A (oxalyl-CoA) decarboxylase was purified from Oxalobacter formigenes by high-pressure liquid chromatography with hydrophobic interaction chromatography, DEAE anion-exchange chromatography, and gel permeation chromatography. The enzyme is made up of four identical subunits (Mr, 65,000) to give the active enzyme (Mr, 260,000). The enzyme catalyzed the thiamine PPi-dependent decarboxylation of oxalyl-CoA to formate and carbon dioxide. Apparent Km and Vmax values, respectively, were 0.24 mM and 0.25 mumol/min for oxalyl-CoA and 1.1 pM and 0.14 mumol/min for thiamine pyrophosphate. The maximum specific activity was 13.5 microM oxalyl-CoA decarboxylated per min per mg of protein.

Acyl Coenzyme A↗