Pyruvamide semicarbazone formation. Kinetics, mechanism, and pertinence to pyruvamide-dependent histidine decarboxylase.
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Amines↗
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Results for “Pyruvate Decarboxylase”
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The assessment of biotin status in man and animals.
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Acetyl-CoA Carboxylase↗
Biotin in clinical medicine--a review.
The recent developments in cofactor therapy of inherited biochemical disease has awakened interest in biotin as a therapeutic agent. This review briefly details the physiological and biochemical aspects of human biotin metabolism. The role of biotin in therapy of human disease is critically examined and the relevant literature extensively reviewed. It is our hope that this review will stimulate further clinical interest in the treatment of biotin-responsive human disorders.
Acetyl-CoA Carboxylase↗
Biotinidase deficiency.
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Amidohydrolases↗
Prenatal treatment of multiple carboxylase deficiency.
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Biotin↗
Pyruvoyl enzymes.
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Adenosylmethionine Decarboxylase↗
Pyruvoyl-dependent histidine decarboxylases. Comparative sequences of cysteinyl peptides of the enzymes from Lactobacillus 30a, Lactobacillus buchneri, and Clostridium perfringens.
The two cysteinyl residues present in histidine decarboxylase from Lactobacillus 30a differ greatly in reactivity. One (class 1) reacts readily in the native state with dithiobis-(2-nitrobenzoate) with complete loss of enzyme activity; the other (class 2) reacts only after denaturation of the enzyme (Lane, R. S., and Snell, E. E. (1976) Biochemistry 15, 4175-4179). These differences in reactivity permitted use of covalent (disulfide) chromatography to isolate separate peptides that contain these two residues. Sequence analysis showed that the class 1 cysteinyl residue is at position 147 in a hydrophilic portion of the alpha chain (Huynh, Q. K., Recsei, P. A., Vaaler, G. L., and Snell, E. E. (1984) J. Biol. Chem. 259, 2833-2839), while the class 2 cysteinyl residue is present at position 71, adjacent to a hydrophobic portion of the same chain. Cysteinyl peptides identical with or homologous to the class 2 cysteinyl peptide of the Lactobacillus 30a enzyme were isolated from the alpha subunits of histidine decarboxylases from Lactobacillus buchneri and Clostridium perfringens, respectively. The L. buchneri enzyme also contained a peptide homologous to the class 1 cysteinyl peptide from Lactobacillus 30a. However, no corresponding peptide was present in the enzyme from C. perfringens, in which the second cysteinyl residue of the alpha chain occupies position 3, very near the essential pyruvoyl residue. This enzyme, unlike those from Lactobacillus 30a or L. buchneri, also contains one cysteinyl residue in its beta chain. Although Cys 147 is an active site residue in histidine decarboxylase from Lactobacillus 30a, the absence of a corresponding residue in the C. perfringens enzyme confirms previous indications (Recsei, P. A., and Snell, E. E. (1982) J. Biol. Chem. 257, 7196-7202) that this SH group is not essential for decarboxylase action.
Amino Acid Sequence↗
Pyruvoyl-dependent histidine decarboxylase from Lactobacillus 30a. Covalent modifications of aspartic acid 191, lysine 155, and the pyruvoyl group.
The pyruvoyl-dependent histidine decarboxylase from Lactobacillus 30a is rapidly inactivated by incubation with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and glycine ethyl ester. On 90% of inactivation, 1.3 residues of [14C]glycine ethyl ester are incorporated per alpha subunit; nearly 60% of this is linked to the beta-carboxyl group of Asp-191. Histamine, a competitive inhibitor, protects against this inactivation. The KM value of the modified enzyme for histidine (6.2 mM) is much higher than that of the unmodified enzyme (KM = 0.4 mM); catalytic activity is reduced but not eliminated. Thus, Asp-191 is the most reactive accessible carboxyl group under these conditions and is close to the substrate-binding site, but apparently is not essential for catalysis. At pH 8.0, fluorodinitrobenzene inactivates histidine decarboxylase completely with the incorporation of two dinitrophenyl residues/alpha subunit; the modified residues are Lys-155 and Cys-228. Urocanic acid, a competitive inhibitor, protects against inactivation. Treatment with mercaptoethanol restores the free -SH of Cys-228 but does not restore activity. Conversion of Cys-228 to its cyano derivative slows but does not prevent dinitrophenylation of Lys-155; the resulting derivative is catalytically inactive. Thus, Lys-155 is located within the active site and may play an essential role in catalysis. Finally, histidine methyl ester was shown to inhibit this decarboxylase by forming a Schiff's base with the essential pyruvoyl group.
Aspartic Acid↗
Purification and biochemical characterization of a pyruvate-specific class II aldolase, HpaI.
HpaI, a class II pyruvate-specific aldolase involved in the catabolic pathway of hydroxyphenylacetate, is overexpressed and purified. A previous suggestion that phosphate is involved in proton transfer of pyruvate, based on the crystal structure of the homologous 2-dehydro-3-deoxygalactarate aldolase, is not substantiated from biochemical studies with HpaI. Thus, specific activities of the enzyme for the substrate 4-hydroxy-2-ketopentanoate in sodium HEPES and Tris-acetate buffers are higher than in sodium phosphate buffer. The enzyme also catalyzed the partial reaction of pyruvate proton exchange with an initial rate of 0.77 mmol min(-)(1) mg(-)(1) in phosphate-free buffer, as monitored by nuclear magnetic resonance. Steady-state kinetic analysis shows that the enzyme is also able to catalyze the aldol cleavage of 4-hydroxy-2-ketohexanoate and 3-deoxy-d-manno-oct-2-ulosonic acid (KDO). The enzyme exhibits significant oxaloacetate decarboxylase activity, with a k(cat) value 2.4-fold higher than the corresponding value for the aldol cleavage of 4-hydroxy-2-ketopentanoate. Sodium oxalate, an analogue of the enolate intermediate of the enzyme-catalyzed reaction, is a competitive inhibitor of the enzyme, with a K(i) value of 5.5 microM. Replacement of an active site arginine residue (R70) with alanine by site-specific mutagenesis resulted in an enzyme that lacks both aldolase and decarboxylase activities. The mutant enzyme is also unable to catalyze pyruvate proton exchange. The dissociation constant for pyruvate in the R70A mutant, determined by fluorescence titration, is similar to that of the wild-type enzyme, indicating that pyruvate binding is not affected by this mutation. Together, the results show that R70 influences catalysis in HpaI, particularly at the pyruvate proton exchange step.
Alanine↗
[Central metabolism in Acinetobacter sp. grown on ethanol].
The ethanol-grown cells of the mutant Acinetobacter sp. strain 1NG, incapable of producing exopolysaccharides, were analyzed for the activity of enzymes of the tricarboxylic acid (TCA) cycle and some biosynthetic pathways. In spite of the presence of both key enzymes (isocitrate lyase and malate synthase) of the glyoxylate cycle, these cells also contained all enzymes of the TCA cycle, which presumably serves biosynthetic functions. This was evident from the high activity of isocitrate dehydrogenase and glutamate dehydrogenase and the low activity of 2-oxoglutarate dehydrogenase. Pyruvate was formed in the reaction catalyzed by oxaloacetate decarboxylase, whereas phosphoenolpyruvate (PEP) was synthesized by the two key enzymes (PEP carboxykinase and PEP synthase) of gluconeogenesis. The proportion between these enzymes was different in the exponential and the stationary growth phases. The addition of the C4-dicarboxylic acid fumarate to the ethanol-containing growth medium led to a 1.5- to 2-fold increase in the activity of enzymes of the glyoxylate cycle, as well as of fumarate hydratase, malate dehydrogenase, PEP synthase, and PEP carboxykinase (the activity of the latter enzyme increased by more than 7.5 times). The data obtained can be used to improve the biotechnology of production of the microbial exopolysaccharide ethapolan on C2-substrates.
Acinetobacter↗
Simultaneous preparation of the three biotin-containing mitochondrial carboxylases from rat liver.
Affinity chromatography on avidin-Sepharose column was used to bind the biotin-containing carboxylases from rat liver. With a biotin gradient (0-0.3 mM), peaks of activity of pyruvate, propionly CoA and beta-methylcrotonyl CoA carboxylases co-eluted. Subsequent separation of the three carboxylases was attained using DEAE-Sepharose chromatography. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed each of the enzymes to be pure, with pyruvate carboxylase giving a single subunit band (Mr 130 000), propionyl-CoA carboxylase giving two bands (Mr 73 000 and 56 500) and beta-methylcrotonyl-CoA carboxylase giving two bands (Mr 75 000 and 60 000). The specific activity of propionyl-CoA carboxylase (15.8 munits/mg) and beta-methylcrotonyl-CoA carboxylase (24.2 munits/mg) were comparable with reported activities for these purified enzymes, while that of pyruvate carboxylase (1.25 munits/mg) was low. This is a suitable method for the simultaneous preparation of purified carboxylases for the specific purpose of raising antisera to these enzymes.
Animals↗
The role of biotin and sodium in the decarboxylation of oxaloacetate by the membrane-bound oxaloacetate decarboxylase from Klebsiella aerogenes.
The biotin-containing oxaloacetate decarboxylase from Klebsiella aerogenes catalyzed the Na+-dependent decarboxylation of oxaloacetate to pyruvate and bicarbonate (or CO2) but not the reversal of this reaction, not even in the presence of an oxaloacetate trapping system. The enzyme catalyzed an avidin-sensitive isotopic exchange between [1-14C]pyruvate and oxaloacetate, which indicated the intermediate formation of a carboxybiotin enzyme. Sodium ions were not required for this partial reaction, but promoted the second partial reaction, the decarboxylation of the carboxybiotin enzyme, thus accounting for the Na+ requirement of the overall reaction. Therefore, the 14CO2-enzyme which was formed upon incubation of the decarboxylase with [4-15C]oxaloacetate, could only be isolated if Na+ ions were excluded. Preincubation of the decarboxylase with avidin also prevented its labelling with 14CO2. The isolated 14CO2-labelled oxaloacetate decarboxylase revealed the following properties. It was slowly decarboxylated at neutral pH and rapidly upon acidification. The 14CO2 residues of the 14CO2-enzyme could be transferred to pyruvate yielding [4-14C]oxaloacetate. In the presence of Na+ this 14CO2 transfer was repressed by the simultaneous decarboxylation of the 14CO2-enzyme. However, Na+ alone was insufficient as a cofactor for the decarboxylation of the isolated 14CO2-enzyme, since this required pyruvate in addition to Na+. It is therefore concluded that the decarboxylation of oxaloacetate proceeds over a CO2-enzyme--pyruvate complex and that free CO2-enzyme is an abortive reaction intermediate. The activation energy of the enzymic decarboxylation of oxaloacetate changed with temperature and was about 113 kJ below 11 degrees C, 60 kJ between 11 degrees C and 31 degrees C and 36 kJ between 31--45 degrees C.
Bacterial Proteins↗
Effect of pyruvate on glucose metabolism in Clostridium acetobutylicum.
Pyruvate effects on the metabolism of Clostridium acetobutylicum during glucose fermentation were studied. After addition to the culture medium, the pyruvate was rapidly used, provoking several changes in the metabolic pattern of the bacteria. When pyruvate addition occurred early in the fermentation, the glucose utilization decreased and the solventogenic phase was not induced. When pyruvate was added during solventogenesis, glucose consumption was slightly affected and the cells fermented both substrates simultaneously: however, the acidogenic phase started again to the detriment of solvent formation. Usually, during the solvent phase, the cells remetabolized acetic and butyric acids into solvents, but when pyruvate was added, the utilization of acids was stopped and the specific rates of acetate and butyrate formation increased immediately. The acidogenic growth phase was characterized by high levels of acetate and butyrate kinase which dropped during the solvent phase. Addition of pyruvate limited the down shift of these two enzymes and the levels of the activities remained constant during the course of the fermentation. Conversely, the acetoacetate decarboxylase, which is characteristic of the solvent phase, decreased sharply in the presence of pyruvate. The fact that the specific rate of glucose consumption was not decreased by the pyruvate metabolism, a cosubstrate, proves that the phosphoroclastic reaction is not a limiting step. Furthermore, the pyruvate utilization represented a promising approach to obtain useful data on the intracellular compounds implicated in the mechanism for switching from the acidogenic to the solventogenic phase.
Clostridium↗
Inactivation and modification of phosphoenolpyruvate carboxykinase differentially labeled with bromopyruvate.
Phosphoenolpyruvate carboxylkinase (GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32) is inactivated by bromopyruvate with specific substrate protection against the inactivation. Despite the fact that the enzyme also is known to possess oxalacetate decarboxylase activity, the modification does not appear to be directed toward a pyruvate or enolpyruvate binding site, as evident from the kinetics of the inactivation and from protection studies. Thus, the reactivity of bromopyruvate is different than toward several other enzymes where pyruvate is a substrate or product. Acetopyruvate and oxalate inhibit carboxykinase activity, but neither of these compounds, nor pyruvate, protects against the inactivation. Using differentially labeled enzyme, it was shown that modification of one sulfhydryl is sufficient to cause loss of both catalytic activities. Protection by inosine nucleotides was found to be similar in each instance. It would appear that a common sulfhydryl is critical to both carboxykinase and oxalacetate decarboxylase activities, and that each utilizes the same nucleotide binding site, despite the known different roles of the nucleotide in each reaction.
Animals↗
Microassay of decarboxylation reactions in cultured cells.
The currently described methods for determination of decarboxylation reaction rates in cultured cells require large quantities of cells and often involve cell manipulation prior to assay. We describe a simple microassay for the rapid measurement of various decarboxylation reaction rates in intact cultured cells. The assay is based on the traditional measurement of 14CO2 generated from 14C-labeled substrates. Key to the method is a novel modification of the standard petri dish. Pyruvate dehydrogenase, branched chain alpha-ketoacid dehydrogenase, alpha-ketoglutarate dehydrogenase, and ornithine decarboxylase activities were determined in adult cardiomyocyte cultures containing only 0.1-0.5 mg of protein per culture dish. Efficiency of 14CO2 collection ranged between 94 and 100%. Pharmacological enhancement or inhibition of pyruvate dehydrogenase activity was easily detected in the culture system. This new method simplifies the measurement of various decarboxylation reaction rates in cultured cells and allows rapid, reproducible measurements to be made on small numbers of cells without perturbation of the culture conditions or the cells themselves.
Animals↗
Bifunctionality and pseudoisozymes of pyruvate kinase from codfish muscle.
Pyruvate kinase has been purified from codfish muscle. The ratio of phosphotransferase and oxalacetate decarboxylase activities remains relatively constant throughout purification steps. These two activities are dependent as well as sensitive to sulfhydryl reagents. In the presence of dithioerythritol, only one molecular form of pyruvate kinase is detected. However, the enzyme exists as four pseudoisozymes in the presence of 2-mercaptoethanol. The pseudoisozymes of codfish pyruvate kinase are interconvertible under the influence of sulfhydryl reagents.
Animals↗
Glycine decarboxylase in Rhodopseudomonas spheroides and in rat liver mitochondria.
1. Glycine decarboxylase and glycine-bicarbonate exchange activities were detected in extracts of Rhodopseudomonas spheroides and in rat liver mitochondria and their properties were studied. 2. The glycine decarboxylase activity from both sources is stimulated when glyoxylate is added to the assay system. 3. Several proteins participate in these reactions and a heat-stable low-molecular-weight protein was purified from both sources. 4. These enzyme activities increase markedly when R. spheroides is grown in the presence of glycine, glyoxylate, glycollate, oxalate or serine. 5. All the enzymes required to catalyse the conversion of glycine into acetyl-CoA via serine and pyruvate were detected in extracts of R. spheroides; of these glycine decarboxylase has the lowest activity. 6. The increase in the activity of glycine decarboxylase on illumination of R. spheroides in a medium containing glycine, and the greater increase when ATP is also present in the medium, probably accounts for the increased incorporation of the methylene carbon atom of glycine into fatty acids found previously under these conditions (Gajdos, Gajdos-Török, Gorchein, Neuberger & Tait, 1968). 7. The results are compared with those obtained by other workers on the glycine decarboxylase and glycine-bicarbonate exchange activities in other systems.
Alcohol Oxidoreductases↗
Use of p-aminobenzoic acid and tritiated cyanoborohydride for the detection of pyruvoyl residues in proteins.
A procedure for the detection of covalently bound pyruvic acid in purified proteins or in crude extracts is described. The dialyzed sample is first treated with sodium cyanoborohydride to reduce any Schiff bases present and then incubated with p-aminobenzoic acid and sodium [3H]cyanoborohydride. Derivatized proteins are visualized by fluorography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel slices containing the labeled proteins are hydrolyzed, and, after removal of polyacrylic acid, the hydrolysate is subjected to ion-exchange high-performance liquid chromatography. The presence of pyruvic acid is established by the detection of a tritiated, 280-nm absorbing compound with a retention time corresponding to that of synthetic N-(p-carboxyphenyl)alanine. The procedure is capable of detecting protein-bound pyruvic acid in the picomolar range and is easily modified to screen for other covalently bound keto acids.
4-Aminobenzoic Acid↗