Dual role of a single multienzyme complex in the oxidative decarboxylation of pyruvate and branched-chain 2-oxo acids in Bacillus subtilis.

The pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase activities of Bacillus subtilis were found to co-purify as a single multienzyme complex. Mutants of B. subtilis with defects in the pyruvate decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) components of the pyruvate dehydrogenase complex were correspondingly affected in branched-chain 2-oxo acid dehydrogenase complex activity. Selective inhibition of the E1 or lipoate acetyltransferase (E2) components in vitro led to parallel losses in pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complex activity. The pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complexes of B. subtilis at the very least share many structural components, and are probably one and the same. The E3 component appeared to be identical for the pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complexes in this organism and to be the product of a single structural gene. Long-chain branched fatty acids are thought to be essential for maintaining membrane fluidity in B. subtilis, and it was observed that the ace (pyruvate dehydrogenase complex) mutant 61142 was unable rapidly to take up acetoacetate, unlike the wild-type, indicative of a defect in membrane permeability. A single pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complex can be seen as an economical means of supplying two different sets of essential metabolites.     

Regulation of valine catabolism in Pseudomonas putida

The activities of six enzymes which take part in the oxidation of valine by Pseudomonas putida were measured under various conditions of growth. The formation of four of the six enzymes was induced by growth on d- or l-valine: d-amino acid dehydrogenase, branched-chain keto acid dehydrogenase, 3-hydroxyisobutyrate dehydrogenase, and methylmalonate semialdehyde dehydrogenase. Branched-chain amino acid transaminase and isobutyryl-CoA dehydrogenase were synthesized constitutively. d-Amino acid dehydrogenase and branched-chain keto acid dehydrogenase were induced during growth on valine, leucine, and isoleucine, and these enzymes were assumed to be common to the metabolism of all three branched-chain amino acids. The segment of the pathway required for oxidation of isobutyrate was induced by growth on isobutyrate or 3-hydroxyisobutyrate without formation of the preceding enzymes. d-Amino acid dehydrogenase was induced by growth on l-alanine without formation of other enzymes required for the catabolism of valine. d-Valine was a more effective inducer of d-amino acid dehydrogenase than was l-valine. Therefore, the valine catabolic pathway was induced in three separate segments: (i) d-amino acid dehydrogenase, (ii) branched-chain keto acid dehydrogenase, and (iii) 3-hydroxyisobutyrate dehydrogenase plus methylmalonate semialdehyde dehydrogenase. In a study of the kinetics of formation of the inducible enzymes, it was found that 3-hydroxyisobutyrate and methylmalonate semialdehyde dehydrogenases were coordinately induced. Induction of enzymes of the valine catabolic pathway was studied in a mutant that had lost the ability to grow on all three branched-chain amino acids. Strain PpM2106 had lowered levels of branched-chain amino acid transaminase and completely lacked branched-chain keto acid dehydrogenase when grown in medium which contained valine. Addition of 2-ketoisovalerate, 2-ketoisocaproate, or 2-keto-3-methylvalerate to the growth medium of strain PpM2106 resulted in induction of normal levels of branched-chain keto acid dehydrogenase; therefore, the branched-chain keto acids were the actual inducers of branched-chain keto acid dehydrogenase.     

Recent Developments in NAD(P)H Regeneration for Enzymatic Reductions in One- and Two-Phase Systems

This review discusses recent achievements in the field of cofactor regeneration for the nicotinamide cofactors NADH and NADPH. The examples discussed include alcohol dehydrogenases, formate dehydrogenase, glucose dehydrogenase and a hydrogenase. For the reaction either one-phase systems or two-phase systems in combination with an organic solvent are discussed. For the enantioselective reduction of 2-octanone to (R)-2-octanol it could be shown that enzyme coupled NADPH regeneration with glucose dehydrogenase and glucose results in shorter reaction times and higher yields when compared to the substrate coupled regeneration with 2-propanol.

ADH: alcohol dehydrogenase; LDH: Lactose dehydrogenase; GDH: Glucose dehydrogenase; FDH: Formate dehydrogenase; LB-ADH: alcohol dehydrogenase from Lactobacillus brevis; HL-ADH: alcohol dehydrogenase from horse liver; TB-ADH: alcohol dehydrogenase from Thermoanaerobicum brockii; PS-GDH: Glucose dehydrogenase from Pseudomonas species; [BMIM][PF₆]: Butyl-methyl-imidazoliumhexafluorophosphate     

TCA循环中间产物对酿酒酵母胞内代谢关键酶活性的影响

对酿酒酵母在添加苹果酸、柠檬酸和琥珀酸的混合培养基与其在YEPD培养基中胞内丙酮酸激酶、葡萄糖-6-磷酸脱氢酶、异柠檬酸脱氢酶、苹果酸脱氢酶、乙醇脱氢酶的酶活力差异进行了对比分析。结果表明:添加苹果酸使胞内丙酮酸激酶、异柠檬酸脱氢酶、苹果酸脱氢酶、乙醇脱氢酶的酶活分别下降34.82%、57.23%、39.15%、12.10%;添加柠檬酸使胞内丙酮酸激酶、异柠檬酸脱氢酶、苹果酸脱氢酶的酶活分别下降50.17%、42.20%、48.40%;添加琥珀酸使胞内丙酮酸激酶、葡萄糖-6-磷酸脱氢酶、异柠檬酸脱氢酶、苹果酸脱氢酶、乙醇脱氢酶的酶活分别下降34.16%、34.16%、50.87%、50.87%、12.37%。丙酮酸激酶、异柠檬酸脱氢酶和苹果酸脱氢酶对3种有机酸的耐受性较差,葡萄糖-6-磷酸脱氢酶、乙醇脱氢酶对3种有机酸的耐受具有选择性。     

Specific modification of a single cysteine residue in both bovine liver glutamate dehydrogenase and yeast glyceraldehyde-3-phosphate dehydrogenase. Difference in the mode of modification by pyrene maleimide

Pyrene maleimide is shown to be a 'half of the sites' reagent for glutamate dehydrogenase and for glyceraldehyde-3-phosphate dehydrogenase. The modified residues are identified as cysteine-115 for glutamate dehydrogenase and cysteine-149 for glyceraldehyde-3-phosphate dehydrogenase. The two enzymes react differently with pyrene maleimide. Whereas the hydrophobic environment of cysteine-115 directs the modification of glutamate dehydrogenase, the high reactivity of cysteine-149 determines the specific modification of glyceraldehyde-3-phosphate dehydrogenase. Glutamate dehydrogenase activity is unaltered by the modification: glyceraldehyde-3-phosphate dehydrogenase activity in inhibited.     

The topology of phosphogluconate dehydrogenases in rat liver microsomes

Rat liver microsomes are known to contain a 6-phosphogluconate dehydrogenase which differs from the 6-phosphogluconate dehydrogenase in the soluble fraction. Microsomes which were washed once bind the soluble phosphogluconate dehydrogenase more tightly than they do glucose-6-phosphate dehydrogenase. Microsomes washed three times in 0.15 M Tris-HCl, pH 8.0, contain only the microsomal 6-phosphogluconate dehydrogenase. Two observations show that this dehydrogenase is located in the cisternae. First, this dehydrogenase is inactive in intact, three times washed microsomes. Second, proteolytic inactivation of 6-phosphogluconate dehydrogenase like that of the cisternal enzyme glucose-6-phosphatase requires disruption of the membrane. Under the conditions used, detergent did not affect the proteolytic inactivation of NADPH-cytochrome c reductase, an enzyme located on the external surface. The excellent correspondence between the activations of hexose phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in microsomes at various stages of disruption of the microsomal membrane produced by detergent supports the earlier contention that these two dehydrogenases are reducing NADP in the same region of the microsomes. A similar experiment which shows an exact correspondence between the activations of 6-phosphogluconate dehydrogenase and mannose-6-phosphatase with increasing concentrations of detergent indicates that the activation of the dehydrogenase can be explained solely by the penetration of the substrates to the active dehydrogenase within the microsomes and strongly suggests that the dehydrogenase is catalytically active in the cisternae.     

Changes in the activity of selected enzymes during starvation of Physarum polycephalum

The levels of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, lactate dehydrogenase, and cyclic phosphodiesterase activities were examined in growing and starving plasmodia of Physarum polycephalum. The activities of lactate dehydrogenase, 6-phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase decreased whereas that of cyclic phosphodiesterase increased. The change in activity of lactate dehydrogenase was the result of the variation of the activity of a single enzyme quite similar to the lactate dehydrogenases of higher animals.     

Biochemical Basis of Obligate Autotrophy in Nitrosomonas europaea

The specific activities of isocitric dehydrogenase, alpha-ketoglutaric dehydrogenase, succinic dehydrogenase, malic dehydrogenase, and reduced nicotinamide adenine dinucleotide (NADH) oxidase were determined in extracts of Nitrosomonas europaea and compared with the corresponding values for Anacystis nidulans and autotrophically grown Hydrogenomonas eutropha. In common with other obligate autotrophs and in contrast to facultative autotrophs, Nitrosomonas extracts lacked alpha-ketoglutaric dehydrogenase and KCN-sensitive NADH oxidase activity and had low succinic dehydrogenase activity. The Nitrosomonas NADH oxidase appeared to be of the peroxidase type.     

Enzyme histochemical observations on the segmentation of the proximal tubules in the kidney of the female rat.

The segmentation of the proximal tubules in the kidney of the female rat was studied by means of enzyme histochemical reactions and the results compared with those observed in male and recently described by Jacobsen and J0rgensen (1973 a). Reactions were performed for the following soluble, coezyme-dependent oxido-reductases: glucose 6-phosphate dehydrogenase, alpha-glycerophosphate dehydrogenase, 3 alpha-hydroxysteroid dehydrogenase, NAD-as well as NADP-dependent isocitrate dehydrogenases, NAD-dependent malate dehydrogenase, NADP-dependent, decarboxylating malate dehydrogenase, uridine diphosphate glucose dehydrogenase. Measures were taken to reduce enzyme diffusion and eliminate interference from tissue tetrazolium reductases. Furthermore, reactions were performed for a number of less soluble or insoluble enzymes: glucose 6-phosphatase, mitochondrial alpha-glycerophosphate dehydrogenase, beta-hydroxybutyrate dehydrogenase, succinate dehydrogenase and tetrazolium reductases. In the proximal tubules of the female rat all enzymes studied--except beta-hydroxybutyrate dehydrogenase--showed segmental differences, most of them clearly revealing three segments. Sex differences were found concerning all enzymes except uridine diphosphate glucose dehydrogenase and NADP-dependent isocitrate dehydrogenase. The most pronounced sex-related differences were seen in the third segment in which part the male rat showed highest activity in respect to tetrazolium reductases, NAD-dependent isocitrate dehydrogenase, succinate dehydrogenase, beta-hydroxybutyrate dehydrogenase, 3 alpha-hydroxysteroid dehydrogenase and glucose 6-phosphate dehydrogenase and the female in respect to glucose 6-phosphatase, alpha-glycerophosphate dehydrogenases, and NADP-dependent, decarboxylating malate dehydrogenase. A few of the enzymes exhibited minor sex differences in the first two segments.     

Chloral hydrate anesthesia alters cerebral enzymes in the rat. A histochemical study

Cerebral forebrain arterioles and neuropil were analyzed histochemically to determine the effects of chloral hydrate anesthesia on key enzymes of aerobic and anaerobic metabolism, as well as the hexose monophosphate shunt in rats. Significant decreases were observed in cytochrome oxidase, and beta-hydroxybutyrate dehydrogenase in arterioles, while glucose-6-phosphate dehydrogenase and isocitric dehydrogenase showed a significant increase and lactate dehydrogenase showed no significant change. In the neuropil, cytochrome oxidase, isocitrate dehydrogenase and glucose-6-phosphate dehydrogenase showed significant increases following chloral hydrate administration, while beta-hydroxybutyrate dehydrogenase and lactate dehydrogenase showed no significant changes. These data suggest that surgical anesthetic levels of chloral hydrate can impair forebrain metabolism which may lead to altered electrophysiological responses.     

Enzyme activity of thiamine pyrophosphate in the rat after oxythiamine administration

Intraperitoneal injection of hydroxythiamine to rats (1 mmol per kg bw) resulted after 2-4 h in a more than 4-fold decrease in the activity of the oxoglutarate dehydrogenase complex, pyruvate dehydrogenase complex and NADP-dependent isocitrate dehydrogenase in adrenal mitochondria. Inhibition of hyaloplasmic transketolase, 6-phosphogluconate dehydrogenase and NADP-dependent malate dehydrogenase occurred later. Based on the correlation of the time course of enzymatic activity in the adrenals and the decreased concentration of 11-hydroxycorticosteroids in the blood the paramount role in the maintenance of the steroidogenesis among thiamine pyrophosphate-containing enzymes is assigned to the oxoglutarate dehydrogenase and pyruvate dehydrogenase complexes.     

Changes in brown-adipose-tissue mitochondrial processes in streptozotocin-diabetes.

Yeast glucose-6-phosphate dehydrogenase was inhibited by low NADPH concentrations in cell-free extracts, and de-inhibited by GSSG; extensive dialysis of the crude extract did not diminish the GSSG effect. Immunoprecipitation of glutathione reductase abolished the de-inhibition of glucose-6-phosphate dehydrogenase by GSSG. Purified glucose-6-phosphate dehydrogenase was inhibited by NADPH but not de-inhibited by GSSG, and upon addition of pure glutathione reductase GSSG completely de-inhibited the glucose-6-phosphate dehydrogenase.     

Bifunctional protein in carrot contains both aspartokinase and homoserine dehydrogenase activities

We have purified homoserine dehydrogenase to homogeneity and subjected polypeptide fragments derived from digests of the protein to amino acid sequencing. The amino acid sequence of homoserine dehydrogenase from carrot (Daucus carota) indicates that in carrot both aspartokinase and homoserine dehydrogenase activities reside on the same protein. Additional evidence that aspartokinase and homoserine dehydrogenase reside on a bifunctional protein is provided by coelution of activities during purification steps and by enzyme-specific gel staining techniques. Highly purified fractions containing aspartokinase activity were stained for aspartokinase activity, homoserine dehydrogenase activity, and protein. These gels confirmed that aspartokinase activity and homoserine dehydrogenase activity were present on the same protein. This arrangement of aspartokinase and homoserine dehydrogenase activities residing on the same protein is also found in Escherichia coli, which has two bifunctional enzymes, aspartokinase I-homoserine dehydrogenase I and aspartokinase II-homoserine dehydrogenase II. The amino acid sequence of the major form of homoserine dehydrogenase from carrot cell suspension cultures most closely resembles that of the E. coli ThrA gene product aspartokinase I-homoserine dehydrogenase I.     

Enhanced steroidogenesis in hen ovary after malonate administration.

The activities of glucose-6-phosphate dehydrogenase, delta 5-3 beta-hydroxysteroid dehydrogenase and succinic dehydrogenase have been studied histochemically in the ovary of normal and malonate treated hens. Following malonate treatment, glucose-6-phosphate dehydrogenase and delta 5-3 beta-hydroxysteroid dehydrogenase showed increased activities while succinic dehydrogenase activity was diminished significantly. The significance of the above changes has been discussed. The ascorbic acid and cholesterol contents of the ovary were determined to get additional information about steroidogenesis in the gland under such treatment.     

Adrenal oxidative enzymes in ox and sheep. The histochemical demonstration of some pathways involved in chromaffin granule synthesis

Summary Catechol amine secretion is achieved by exocytosis. In this, ATP and protein are also lost from the chromaffin cells. Histochemically various specific coenzyme linked dehydrogenases associated with ATP production have been demonstrated in the adrenals of ox and sheep. These included cytochrome oxidase, DPN and TPN diaphorases, isocitric dehydrogenase, malate dehydrogenase, glutamate dehydrogenase, succinic dehydrogenase, lactic dehydrogenase, alcohol dehydrogenase, -glycerophosphate dehydrogenase and -hydroxybutyrate dehydrogenase. Enzymes of the pentose shunt were found histochemically and biochemically. The RNA content of the adrenal medulla and cortex was also investigated.     

Thermodynamic resolution of cytochrome P450 and characterization of an iron-sulfur center in rat liver microsomal preparations.

Binding of 8-anilinonaphthalene sulfonate (ANS) to glutamate dehydrogenase results in enzyme inhibition and a marked increase in the fluorescence of ANS. Perphenazine and GTP increase the fluorescence of ANS-glutamate dehydrogenase secondary to their known ability to alter the conformation of this enzyme. Aspartate aminotransferases, which form enzyme-enzyme complexes with glutamate dehydrogenase, produce a slight decrease in the fluorescence of ANS-glutamate dehydrogenase.While ANS and perphenazine are allosteric inhibitors of reactions catalyzed by free glutamate dehydrogenase, they do not inhibit reactions catalyzed by aminotransferaseglutamate dehydrogenase complexes. This is in spite of the fact that the aminotransferase does not prevent either ANS or perphenazine from being bound to glutamate dehydrogenase. Therefore, reactions catalyzed by the enzyme-enzyme complex are apparently not inhibited by ANS or perphenazine because binding of the aminotransferase to glutamate dehydrogenase prevents these ligands from altering the conformation of glutamate dehydrogenase. This is consistent with the fact that the aminotransferase also prevents perphenazine from enhancing the fluorescence of ANS-glutamate dehydrogenase.Reactions catalyzed by the enzyme-enzyme complex are inhibited by GTP and the aminotransferase does not prevent GTP from enhancing the fluorescence of ANS-glutamate dehydrogenase. Therefore, binding of the aminotransferase to glutamate dehydrogenase does not prevent GTP from altering the conformation of glutamate dehydrogenase.The fact that the aminotransferase completely prevents perphenazine from increasing the fluorescence of ANS-glutamate dehydrogenase suggests that in the enzymeenzyme complex each glutamate dehydrogenase polypeptide chain can be bound to an aminotransferase polypeptide chain. This would mean that three aminotransferase molecules can be bound to each monomeric unit (M_r 3 × 10⁵) of glutamate dehydrogenase.     

Acetate catabolism in the dissimilatory iron-reducing isolate GS-15.

Acetate-grown GS-15 whole-cell suspensions were disrupted with detergent and assayed for enzymes associated with acetate catabolism. Carbon monoxide dehydrogenase and formate dehydrogenase were not observed in GS-15. Catabolic levels of acetokinase and phosphotransacetylase were observed. Enzyme activities of the citric acid cycle, i.e., isocitrate dehydrogenase, 2-oxoglutarate sythase, succinate dehydrogenase, fumarase, and malate dehydrogenase, were observed.     

Plagiorchis elegans: Histochemical localization of dehydrogenases in the cercarial stage

Cercariae of Plagiorchis elegans Rudolphi 1802 collected from experimentally infected snails, Lymnaea palustris, were subjected to various histochemical tests for dehydrogenase systems. A high degree of activity was demonstrated for succinic dehydrogenase (EC 1.3.99.1), malic dehydrogenase (EC 1.1.1.37), isocitric dehydrogenase (EC 1.1.1.41), α-glycerophosphate dehydrogenase (EC 1.1.1.8), and glucose 6-phosphate dehydrogenase (EC 1.1.1.49). These enzymes were present in the tegument, tail, caudal pocket, excretory bladder, acetabulum, and oral sucker, particularly in the muscles around the stylet. Only moderate activity was obtained for lactic dehydrogenase (EC 1.1.1.27) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) at these sites, glutamic dehydrogenase (EC 1.4.1.2) was localized only in the tails of the cercariae and tests for alcohol dehydrogenase (EC 1.1.1.1) were completely negative. The cerebral ganglia and its commissures stained intensely in the tests for succinic, isocitric, α-glycerophosphate, and glucose 6-phosphate dehydrogenase systems. The results indicate the possibility that several energy-producing sequences may be available to these cercariae.     

The catabolism of branched-chain amino acids occurs via 2-oxoacid dehydrogenase in Saccharomyces cerevisiae.

Saccharomyces cerevisiae possesses 2-oxoacid dehydrogenase (EC 1.2.4.4) similar to that found in mammalian cells. The activity is readily detected in cells which have been cultured in a minimal medium containing a branched-chain amino acid. Mutants defective in lipoamide dehydrogenase also lack 2-oxoacid dehydrogenase and are thus unable to catabolize branched-chain amino acids: 2-oxoacids accumulate in the cultures of these cells. The 2-oxoacid dehydrogenase activity is distinct from both 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase, because it could not be detected in assay conditions which permitted the measurement of 2-oxoglutarate dehydrogenase and vice versa. In addition, a strain lacking 2-oxoglutarate dehydrogenase (kgd1::URA3) retained 2-oxoacid dehydrogenase as did a mutant specifically lacking pyruvate dehydrogenase (pda1::Tn5ble). In complex media the specific activity of this enzyme is highest in YEP (yeast extract-peptone)-glycerol and lowest in YEP-acetate and YEP-fructose. 2-Oxoacid dehydrogenase could not be detected in cells which had been transferred to sporulation medium. These results suggest that in S. cerevisiae the catabolism of branched-chain amino acids occurs via 2-oxoacid dehydrogenase, not via the 'Ehrlich Pathway'.