A mutant of Saccharomyces cerevisiae lacking catabolic NAD-specific glutamate dehydrogenase Growth characteristics of the mutant and regulation of enzyme synthesis in the wild-type strain

NAD-specific glutamate dehydrogenase (GDH-B)¹ was induced in a wild-type strain derived of - 1278b by -amino acids, the nitrogen of which according to known degradative pathways is transferred to 2-oxoglutarate. A recessive mutant (gdhB) devoid of GDH-B activity grew more slowly than the wild type if one of these amino acids was the sole source of nitrogen. Addition of ammonium chloride, glutamine, asparagine or serine to growth media with inducing -amino acids as the main nitrogen source increased the growth rate of the gdhB mutant to the wild-type level and repressed GDH-B synthesis in the wild type. Arginine, urea and allantoin similarly increased the growth rate of the gdhB mutant and repressed GDH-B synthesis in the presence of glutamate, but not in the presence of aspartate, alanine or proline as the main nitrogen source. These observations are consistent with the view that GDH-B in vivo deaminates glutamate. Ammonium ions are required for the biosynthesis of glutamine, asparagine, arginine, histidine and purine and pyrimidine bases. Aspartate and alanine apparently are more potent inducers of GDH-B than glutamate.Anabolic NADP-specific glutamate dehydrogenase (GDH-A) can not fulfil the function of GDH-B in the gdhB mutant. This is concluded from the equal growth rates in glutamate, aspartate and proline media as observed with a gdhB mutant and with a gdhA, gdhB double mutant in which both glutamate dehydrogenases are lacking. The double mutant showed an anomalous growth behaviour, growth rates on several nitrogen sources being unexpectedly low.The following abbreviations and symbols are used GDH-A NADP-specific glutamate dehydrogenase [l-glutamate - NADP⁺ oxido-reductase (deaminating), EC 1.4.1.4] - gdhA genotype associated with GDH-A deficiency - GDH-B NAD-specific glutamate dehydrogenase, [L-glutamate NAD⁺ oxido-reductase (deaminating), EC 1.4.1.2] - gdhB genotype associated with GDH-B deficiency - gdhCR genotype associated with derepressed GDH-B synthesis - specific growth rate (h^-1) - x cell density - t time (h)     

THE MITOCHONDRIAL REDOX STATE OF RAT BRAIN

The use of the glutamate dehydrogenase (EC 1.4.1.3) and β-hydroxybutyrate dehydrogenase (EC 1.1.1.30) reactions for the calculation of the mitochondrial redox state of brain has been examined. To prevent post-mortem anoxic metabolism, brains were frozen in less than a second by using a new technique. Levels of ketone bodies in brain were so low relative to the contamination by blood and extracellular fluid that calculation of the mitochondrial redox state using the β-hydroxybutyrate dehydrogenase reaction was not practical. The concentrations of the non-nucleotide substrates of the glutamate dehydrogenase reaction could be accurately measured in brain and themitochondrial [NAD⁺]/[NADH] ratio calculated from the ratio [α-oxoglutarate] [NH₄⁺]/[glutamate]. The calculation is valid if the ratio [α-oxoglutarate] [NH₄⁺]/[glutamate] in mitochondria is the same as that measured in whole tissue. The evidence supporting this conclusion is the near-equilibrium of the aspartate aminotransferase (EC 2.6.1.l) reaction in brain and the observation by others that the distribution of label between α-oxoglutarate and glutamate in brain, after administration of labelled precursors, conforms to expectation. The alanine aminotransferase (EC 2.6.1.2) reaction was not near equilibrium in brain, probably because of the low in vivo activity of the enzyme.     

2-Oxoglutarate transport: A protential mechanism for regulating glutamate and tricarboxylic acid cycle intermediates in neurons

2-Oxoglutarate (-ketoglutarate) is transported into synaptosomal and synaptoneurosomal preparations by a Na⁺-dependent, high-affinity process that exhibits complex kinetics, and is differentially modulated by glutamate, glutamine, aspartate, malate, and a soluble, heat-labile substance of high molecular weight present in rat brain extracts. Glutamate and aspartate generally inhibit 2-oxoglutarate uptake, but under certain conditions may increase uptake. Glutamine generally increases 2-oxoglutarate uptake, but under certain conditions may inhibit uptake. One interpretation of our results is that 2-oxoglutarate uptake is mediated primarily by a transporter that exhibits negative cooperativity and possesses three regulatory sites that differentially modulate substrate affinity, V_max, and negative cooperativity. Glutamate, aspartate, malate, and 2-oxoglutarate itself may interact with a site that reduces substrate affinity; whereas glutamine, and possibly glutamate and aspartate, appear to interact with another site that increases V_max. A putative regulatory protein appears to abolish negative cooperativity and increases substrate affinity in the absence of glutamine. Based on the evidence that glutamatergic and GABAergic neurons depend on astrocytes to supply precursors to replenish their neurotransmitter and tricarboxylic acid cycle pools, the uptake of 2-oxoglutarate, presumably into synaptic terminals, may reflect a role for this metabolite in replenishing the transmitter and tricarboxylic acid pools, and a role for the transporter as a site at which these pools are regulated.Abbreviations used AAT aspartate aminotransferase - glu glutamate - gln glutamine - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - LDS low-density synaptosomes - OAA oxaloacetate - 2-OG 2-oxoglutarate (-ketoglutarate) - PC pyruvate carboxylase - PDH pyruvate dehydrogenase - TCA tricarboxylic acid Special issue dedicated to Dr. Claude Baxter.     

Glutamate as a precursor of GABA in rat brain and peripheral tissues

Summary The formation of GABA from L-glutamate was investigated in homogenates of rat brain, liver, and kidney, using highly purified [¹⁴C]-L-glutamic acid as substrate and a thin-layer chromatographic separation of products. In agreement with other workers, liberation of [¹⁴C]-CO₂ was found to be stoichiometric with GABA formation in brain homogenates, but not in liver or kidney extracts. Subcellular fractionation and dialysis experiments suggested that most of the GABA synthesis in these peripheral tissues, unlike brain, does not occur via a direct decarboxylation of glutamate and requires one or more cofactors other than pyridoxal phosphate. NAD stimulated GABA formation in dialyzed extracts, and inhibition of GABA-transaminase, bothin vitro andin vivo, caused marked inhibition of GABA formation from glutamate in peripheral extracts. Although a very low GAD activity in liver and kidney cannot be excluded, these experiments suggest a major pathway from glutamate to GABA in these homogenates which includes (1) conversion of glutamate to -ketoglutarate by glutamate dehydrogenase or transaminases, (2) conversion of -ketoglutarate to succinic semialdehyde, and (3) formation of GABA from succinic semialdehyde and glutamate by GABA-transaminase.     

Ethylene biosynthesis in Fusarium oxysporum f. sp. tulipae proceeds from glutamate/2-oxoglutarate and requires oxygen and ferrous ions in vivo

Liquid cultures of the deuteromycete, Fusarium oxysporum f. sp. tulipae, a tulip pathogen, produced high amounts of ethylene during stationary phase. 1-Aminocyclopropane-1-carboxylic acid, the direct precursor of ethylene in plants, was not present in the fungus. Radioactivity from [3,4-³H]glutamate as well as [U-¹⁴C]glutamate was incorporated into ethylene, indicating that it was derived from C3 and C4 of glutamate or 2-oxoglutarate. Ferrous ions markedly stimulated the rate of ethylene formation in vivo, whereas Fe³⁺, Cu²⁺ or Zn²⁺ had little or no effect. Ethylene biosynthesis was strongly inhibited by the heavy metal chelator ,-dipyridine. The effect of ,-dipyridine was fully reversed by Fe²⁺ ions and partially by Cu²⁺ and Zn²⁺ ions but not by the supply of glutamate or 2-oxoglutarate, suggesting that a step in the ethylene biosynthetic pathway downstream of 2-oxoglutarate is dependent on Fe²⁺. When stationary phase cultures were supplied with arginine, ornithine, or proline, ethylene production increased dramatically while addition of glutamate or 2-oxoglutarate had little effect. Tracer studies were performed to test the possibility that an intermediate in the catabolism of arginine to glutamate was the direct precursor of ethylene. In cultures supplied with [U-¹⁴C]arginine or [U-¹⁴C]glutamate, the specific radioactivity of ethylene was closely similar to the specific radioactivity of the endogenous glutamate pool, indicating that glutamate was on the pathway between arginine and ethylene. An enzyme system converting 2-oxoglutarate to ethylene in a reaction dependent on oxygen, ferrous ions and arginine has previously been described in extracts from Penicillium digitatum (Fukuda et al. 1986). The present results suggest that a similar enzyme system catalyzes the final step of ethylene biosynthesis in F. oxysporum.Non-standard abbreviations AdoMet S-adenosyl methionine - ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene forming enzyme     

Utilization of l-alanine by Rhodopseudomonas acidophila

Rhodopseudomonas acidophila strain 7050 can satisfy all its nitrogen and carbon requirements from l-alanine. Addition of 100 M methionine sulfoximine to alanine grown cultures had no effect on growth rate indicating that deamination of alanine via alanine dehydrogenase and re-assimilation of the released NH ₄ ⁺ by glutamine synthetase/glutamate synthase was an insignificant route of nitrogen transfer in this bacterium. Determination of aminotransferase activities in cell-free extracts failed to demonstrate the presence of direct routes from alanine to either aspartate or glutamate. The only active aminotransferase involving l-alanine was the alanine-glyoxylate enzyme (114–167 nmol·min^–1·mg^–1 protein) which produced glycine as end-product. The amino group of glycine was further transaminated to yield aspartate via a glycineoxaloacetate aminotransferase (117–136 nmol·min^–1 ·mg^–1 protein). No activity was observed when 2-oxoglutarate was substituted for oxaloacetate. The formation of glutamate from aspartate was catalysed by aspartate-2-oxoglutarate aminotransferase (85–107 nmol·min^–1·mg^–1 protein). Determinations of free intracellular amino acid pools in alanine and alanine+100 M methionine sulfoximine grown cells showed the predominance of glutamate, glycine and aspartate, providing further evidence that in alanine grown cultures R. acidophila satisfies its nitrogen requirements for balanced growth by transamination.Abbreviations ADH alanine dehydrogenase - GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase - MSO methionine sulfoximine - GOT glutamate-oxaloacetate aminotransferase - GPT glutamate-pyruvate amino-transferase - AGAT alanine-glyoxylate aminotransferase - GOAT glycine-oxaloacetate aminotransferase - GOTAT glycine-2-oxoglutarate aminotransferase - AOAT alanine-oxaloacetate aminotransferase     

Ammonium assimilation in an Arthrobacter sp. “fluorescens”

Ammonium assimilation was studied in a nitrogenfixing Arthrobacter strain grown in both batch and ammonium-limited continuous cultures. Arthrobacter sp. fluorescens grown in nitrogen-free medium or at low ammonium levels assimilated NH ₄ ⁺ via the glutamine synthetase/glutamate synthase pathway. When ammonium was in excess it was assimilated via the alanine dehydrogenase pathway. Very low levels of glutamate dehydrogenase were found, irrespective of growth conditions.Abbreviations GS glutamine synthetase - GOGAT glutamine oxoglutarate aminotransferase - GDH glutamate dehydrogenase - ADH alanine dehydrogenase - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase     

4-aminobutyrate is not available to bacteroids of cowpea Rhizobium MNF2030 in snake bean nodules

Laboratory cultures of cowpea Rhizobium MNF2030 grew on 4-aminobutyrate (GABA) as sole source of carbon and nitrogen. GABA transport was active since it was inhibited by carbonyl cyanide mchlorophenyl hydrazone and 2,4-dinitrophenol and cells developed a 400-fold concentration gradient across the cell membrane. Arsenite treatment of GABA-grown cells revealed stoichiometric conversion of GABA to pyruvate, indicating that 2-oxoglutarate is not an intermediate in GABA catabolism. GABA catabolism by cells of strain MNF2030 grown on GABA appreared to involve GABA transaminase, succinic semialdehyde dehydrogenase and malic enzyme; the first two enzymes were specifically induced by growth on GABA. The deamination process and removal of NH₃ in cells catabolizing GABA involved GABA: 2-oxoglutarate transaminase; glutamate: oxaloacetate aminotransferase; asparate: pyruvate aminotransferase and alanine dehydrogenase.Isolated snakebean bacteroids of strain MNF2030 transported only small amounts of GABA and had uninduced levels of GABA catabolic enzymes, even though the nodules contained significant levels of GABA. The data suggest that GABA is not available to snakebean nodule bacteroids, presumably because of a control imposed by the peribacteroid membrane.Abbreviations CCCP Carbonyl cyanide m-chlorophenyl hydrazone - HEPES N-hydroxyethylpiperazine-N-2-ethanesulphonic acid - DTT dithiothreitol - SSAD succinic semialdehyde dehydrogenase - GABAT 4-aminobutyrate transaminase - GABA 4-aminobutyrate     

Anaerobic energy metabolism of goldfish,Carassius auratus (L.)

Summary The concentrations of pyruvate, lactate, oxalo-acetate, aceto-acetate -hydroxybutyrate, -ketoglutarate, glutamate, NH ₄ ⁺ , NAD⁺ and NADH were measured in goldfish tissues after previous conditioning to normal and anoxic (12h) conditions. For 11 different metabolites efficiency of different extraction methods was tested by means of internal standards. The recoveries were generally over 80%. The substrate/product couples of the reactions catalysed by lactate dehydrogenase, malate dehydrogenase, -hydroxybutyrate dehydrogenase and glutamate dehydrogenase were used as redox parameters. In the lateral red muscle the redox state did not change during 12 h of anoxia. In the dorsal white muscle only the cytoplasmic redox state underwent a change, as indicated by the increase of the lactate/pyruvate ratio from 20 to 110. In liver both cytoplasm and mitochondria were reduced during anoxia. From the measured values the NAD⁺/NADH ratio was found to change only in white muscle, while the calculated free NAD⁺/NADH ratios were reduced in anoxic white muscle cytoplasm, anoxic liver mitochondria, and anoxic liver cytoplasm. Oxalo-acetate concentrations calculated from the equilibrium constants of lactate dehydrogenase and malate dehydrogenase were at least one order of magnitude smaller than the measured values. The data obtained from anoxic goldfish are in contrast to available data on other animals and support earlier reports which indicate that this animal has a special anaerobic metabolism. The results are discussed especially with respect to the role of ethanol as a sink for reducing equivalents.Abbreviations LDH lactate dehydrogenase - MDH malate dehydrogenase - HBDH -hydroxybutyrate dehydrogenase - GIDH glutamate dehydrogenase     

Production of protease with Bacillus licheniformis mutants insensitive to repression of exoenzyme biosynthesis

Two mutant strains of Bacillus licheniformis insensitive to catabolite repression were selected by classical mutagenesis in connection with the development of a fed-batch procedure for protease production. B. licheniformis 4a produced up to 20 U (Anson-Units) subtilisin Carlsberg/ml in fed-batch experiments in the presence of up to 1.5 m glycerol, but was inhibited by excess ammonium. Formation of spores, excretion of -amylase and the biosynthesis of citrate synthase and isocitrate dehydrogenase were likewise not repressed by glycerol. The strain was characterized by unusually low activity of the -oxoglutarate dehydrogenase complex and increased biosynthesis of polyglutamic acid in the presence and excretion of -oxoglutarate in the absence of ammonium, respectively. The results are discussed in view of a possible connection between the defect in the -oxoglutarate dehydrogenase complex and insensitivity to catabolite repression. The second strain B. licheniformis 114 was able to synthesize 11.5 U protease/ml independently of the glycerol and ammonium concentration in the medium. Correspondence to: G. Bierbaum     

Purification and some properties of NAD+-dependent glutamate dehydrogenase from Halobacterium halobium

• 1.1. A NAD⁺-dependent glutamate dehydrogenase (EC 1.4.1.2.) was purified 126-fold from Halobacterium halobium.
• 2.2. Activity and stability of the enzyme were affected by salt concentration. Maximum activity of the NADH-dependent reductive amination of 2-oxoglutarate occurs at 3.2 M NaCl and 0.8 M KCl, and the NAD⁺-dependent oxidative deamination of l-glutamate occurs at 0.9 M NaCl and 0.4 M KCl. The maximum activity is higher with Na⁺ than with K⁺ in the amination reaction while the reverse is true in the deamination reaction.
• 3.3. The apparent K_m values of the various substrates and coenzymes under optimal conditions were: 2-oxoglutarate, 20.2 mM; ammonium, 0.45 M; NADH, 0.07 mM; l-glutamate, 4.0 mM; NAD⁺, 0.30 mM.
• 4.4. No effect of ADP or GTP on the enzyme activity was found. The purified enzyme was activated by some l-amino acids.

     

Glutamate dehydrogenase of the unicellular green alga Scenedesmus acutus

The coenzyme-non-specific glutamate dehydrogenase (EC 1.4.1.3) from Scenedesmus acutus in inhibited by p-hydroxymercuribenzoate only in the deamination reaction. From this result and from its stability in the presence of urea it is concluded that this enzyme exhibits and equilibrium between three conformations: aminating and deaminating conformations induced by NADH-2-oxoglutarate and NAD⁺-glutamate, respectively, and the “native” conformation in the absence of substrates.     

Pullulanase,an enzyme of starch catabolism,is associated with the outer membrane of Klebsiella

Late-log phase cells of Klebsiella sp. 5246 could be converted into spheroplasts with a yield of better than 90% by ethylenediamine tetraacetate/lysozyme treatment in osmotically stabilizing media. Membrane fragments obtained after ultrasonication of spheroplasts were separated by centrifugation to sedimentation equilibrium on a sucrose density gradient. A light membrane fraction with a buoyant density of 1.17±0.02g/cm³ was sought and found to contain the enzymes NADH oxidase, succinate dehydrogenase and D-lactate dehydrogenase. A heavy membrane fraction having a buoyant density of 1.23 ±0.01g/cm³ was characterized by phospholipase A₁ activity and lipopolysaccharide content. By analogy to other gram-negative bacteria, the light and the heavy fraction were assigned, respectively, to the cytoplasmic and the outer membrane of Klebsiella sp. 5246.The organism produced pullulanase in a cellbound form during the exponential phase of growth on soluble starch. Pullulanase was localized exclusively on the outer membrane. Pullulanase is the second protein of the outer membrane with defined enzyme function to become known among gram-negative bacteria, the other one being phospholipase A₁.What had been inferred from physiological studies of growth characteristics on various carbon sources can now be proven directly: Pullulanase implicated in the utilization of branched -glucans in Klebsiella is capable of acting on macromolecular substrates in the environment of the cell by virtue of its association with the outer membrane.Non-Standard Abbreviations EDTA ethylenediamine tetraacetate - SDS sodium dodecyl sulphate - OD optical density List of Enzymes EC 3.2.1. 23 -galactosidase or -D-galactoside galactohydrolase - EC 1.1.1.28 D-lactate dehydrogenase or D-lactate: NAD⁺ oxidoreductase - EC 3.2.1.17 lysozyme or mucopeptide N-acetylmuramoylhydrolase - EC 2.4.1.1 maltodextrin phosphorylase or 1,4--D-glucan: orthophosphate -glucosyltransferase - EC 1.6.99.3 NADH oxidase or NADH: (acceptor) oxidoreductase - EC 3.1.1.32 phospholipase A₁ or phosphatide 1-acylhydrolase - EC 3.2.1.41 pullulanase or pullulan 6-glucanohydrolase - EC 1.3.99.1 succinate dehydrogenase or succinate: (acceptor) oxidoreductase     

Gaba shunt in developing soybean seeds is associated with hypoxia

In the present study we investigated the proposal that the γ-aminobutyrate (Gaba) shunt in developing soybean (Glycine max [L.] Merr.) seeds is associated with hypoxia. The ontogeny and pH profile of enzymes associated with glutamate metabolism (glutamate decarboxylase [EC 4.1.1.15]. Gaba transaminase [EC 2.6.1.19], succinic semialdehyde dehydrogenase [EC 1.2.1.16], glutamate dehydrogenase [EC 1.4.1.2], glutamate:oxaloacetate transaminase [EC 2.6.1.1], glutamate:pyruvate transaminase [EC 2.6.1.2] and 2-oxoglutarate dehydrogenase complex [EC 1.2.4.2]) and hypoxia (alcohol dehydrogenase [ADH, EC 1.1.1.1] and pyruvate decarboxylase [PDC, EC 4.1.1.1]) were determined in cotyledons, nucellus and seed-coat tissues. Gaba-shunt enzymes were ubiquitous in the developing seed. Activities of enzymes catalyzing glutamate-C entry into the Krebs cycle via 2-oxoglutarate were generally greater than those of Gaba-shunt enzymes. In cotyledons, the activity of ADH increased throughout seed development (up to 72 days after anthesis [DAA]), whereas PDC was static during early development, then increased. In contrast, the activities of ADH and PDC in maternal tissues (nucellus and seed coat) were initially high, then declined dramatically after 37 DAA. The adenylate energy charge (AEC) = ([ATP]+0.5 [ADP])/ ([ATP] + [ADP] + [AMP]) of soybean seeds from fruits (37 DAA) frozen in situ was low (0.67±0.01) compared to the AEC of adjacent pod tissue (0.82 ± 0.04) and cotyledons exposed to air (0.84 ± 0.01). A 60-min time-course study showed that the rate of [U-¹⁴C]-glutamate catabolism by an intact excised cotyledon at 37 DAA was markedly lower at 8 and 0% O₂ than at 21%; the pool size of [¹⁴C]-Gaba was unaffected. The data indicated that: (1) Gaba-shunt activity is not a response to limited glutamate deamination/transamination: (2) the soybean seed is hypoxic; and (3) the relative partitioning of glutamate-C through glutamate decarboxylase is increased by hypoxia.     

Degradation of α-amylase fromAspergillus oryzae with firmly bound proteinases at pH 3.0

Incubation of highly purified -amylase fromAspergillus oryzae (EC 3.2.1.1) with 0.01M acetate buffer, pH 3.0, resulted in degradation of the -amylase. The molecular weight values of degradation products were 42 K, 37 K, and 28 K. Incubation of the purified -amylase in 0.02m phosphate buffer, pH 7.5, at 30°C for 17 h, however, resulted in no degradation of the -amylase molecule.Incubation of the purified -amylase with proangiotensin at pH 3.0 for 24 h resulted in cleavage of Tyr⁴-Ile⁵, His⁶-Pro⁷, Pro⁷-Phe⁸, Phe⁸-His⁹, and His⁹-Leu¹⁰. Thus, it appears that proteolytic activities firmly bound to -amylase are identical withAspergillus aspartic proteinase (EC 3.4.23.6) andAspergillus acid carboxypeptidase (EC 3.4.16.1).     

Microbial production of l-leucine from α-ketoisocaproate by Corynebacterium glutamicum

Summary A process for l-leucine production was studied using Corynebacterium glutamicum for the conversion of -ketoisocaproate. When this precursor was added to the culture medium in a concentration of 20 g/l about 16 g/l l-leucine were formed after a fermentation time of 57 h and the molar yield was 91%. Using a fed-batch culture it was possible to produce 24 g/l of l-leucine from 32 g/l of -ketoisocaproate within 23 h. Enzymatic studies indicate that in this glutamate-producing bacterium -ketoisocaproate is converted into l-leucine via the transaminase B reaction and l-glutamate is regenerated by the glutamate dehydrogenase. By the addition of -ketoisocaproate to the culture medium the specific activity of transaminase B was increased threefold.     

Acetyl coenzyme A and coenzyme A contents of growing Clostridium kluyveri as determined by isotope assays

CoASH and some of its acyl derivatives, especially acetyl-SCoA, occupy a central position in the energy metabolism of the anaerobic Clostridium kluyveri, both as intermediates and as regulatory effectors. The steady state concentrations of these compounds were determined in growing cultures of this organism using an anaerobic and fast deproteinization technique and radio isotope assays. Acetyl-SCoA was determined as [1-¹⁴C]citrate formed in the presence of [4-¹⁴C]oxaloacetate and citrate synthase; 0.49 mol/g cell wet wt. were found CoASH, CoAS-SCoA after borohydride reduction, and total acyl derivatives of coenzyme A after hydrolysis of the thiol esters were converted to thioethers with [2,3-¹⁴C]N-ethylmaleimide and brought to radiochemical purity by chromatographic methods. While disulfides of coenzyme A were undetectable, 0.13 mol CoASH and 1.17 mol of total acyl-SCoA per g wet wt. were found. These data are consistent with the regulatory scheme of the energy metabolism of C. kluyveri previously proposed.Abbreviations DTE dithioerythritol - NEM N-ethylmaleimide - NES N-ethylsuccinimide Enzymes (EC 2.7.2.1) Acetate kinase, ATP: acetate phosphotransferase - (EC 3.1.3.1) Alkaline phosphatase, orthophosphoric monoester phosphohydrolase - (GOT) Aspartate aminotransferase - (EC 2.6.1.1) L-aspartate:2-oxoglutarate aminotransferase - (CS) Citrate synthase - (EC 4.1.3.7) citrate oxaloacetate-lyase (pro 3S-CH₂COOacetyl-CoA) - (EC 2.8.3.8) CoA-transferase, acyl-CoA:acetate CoA-transferase - (EC 1.1.1.37) Malate dehydrogenase, L-malate:NAD⁺ oxidoreductase - (EC 1.18.1.3) NADH:ferredoxin reductase, ferredoxin:NAD⁺ oxidoreductase - (EC 3.1.4.1) Phosphodiesterase (snake venom), orthophosphoric diester phosphohydrolase - (EC 2.3.1.8) Phosphotransacetylase, acetyl-CoA:orthophosphate acetyltransferase - (EC 2.3.1.9) Thiolase, acetyl-CoA:acetyl-CoA C-acetyltransferase A preliminary account of this work has been given (Decker et al. 1976)     

Biochemical Characterization of the Yeast Yarrowia lipolytica Overproducing Carboxylic Acids from Ethanol: Nitrogen Metabolism Enzymes

A comparative assay of nitrogen metabolism enzymes in the Yarrowia lipolytica mutant N1 grown under conditions promoting the overproduction of either -ketoglutaric acid (KGA) or citric acid showed that the overproduction of KGA correlates with an increase in the activities of the NAD- and NADP-linked glutamate dehydrogenase, glutamic–pyruvic transaminase, and glutamic–oxaloacetic transaminase reactions. These reactions are likely to be responsible for the overproduction of KGA by this mutant. In contrast, the overproduction of citric acid correlated with a decline in the activities of the NAD- and NADP-linked glutamate dehydrogenases and with an increase in the activities of glutamine synthetase and glutamate synthase.     

The kinetics of enzyme changes in yeast under conditions that cause the loss of mitochondria

1. Aerobically grown yeast having a high activity of glyoxylate-cycle, citric acid-cycle and electron-transport enzymes was transferred to a medium containing 10% glucose. After a lag phase of 30min. the yeast grew exponentially with a mean generation time of 94min. 2. The enzymes malate dehydrogenase, isocitrate lyase, succinate–cytochrome c oxidoreductase and NADH–cytochrome c oxidoreductase lost 45%, 17%, 27% and 46% of their activity respectively during the lag phase. 3. When growth commenced pyruvate kinase, pyruvate decarboxylase, alcohol dehydrogenase, glutamate dehydrogenase (NADP⁺-linked) and NADPH–cytochrome c oxidoreductase increased in activity, whereas aconitase, isocitrate dehydrogenase (NAD⁺- and NADP⁺-linked), α-oxoglutarate dehydrogenase, fumarase, malate dehydrogenase, succinate–cytochrome c oxidoreductase, NADH–cytochrome c oxidoreductase, NADH oxidase, NADPH oxidase, cytochrome c oxidase, glutamate dehydrogenase (NAD⁺-linked), glutamate–oxaloacetate transaminase, isocitrate lyase and glucose 6-phosphate dehydrogenase decreased. 4. During the early stages of growth the loss of activity of aconitase, α-oxoglutarate dehydrogenase, fumarase and glucose 6-phosphate dehydrogenase could be accounted for by dilution by cell division. The lower rate of loss of activity of isocitrate dehydrogenase (NAD⁺- and NADP⁺-linked), glutamate dehydrogenase (NAD⁺-linked), glutamate–oxaloacetate transaminase, NADPH oxidase and cytochrome c oxidase implies their continued synthesis, whereas the higher rate of loss of activity of malate dehydrogenase, isocitrate lyase, succinate–cytochrome c oxidoreductase, NADH–cytochrome c oxidoreductase and NADH oxidase means that these enzymes were actively removed. 5. The mechanisms of selective removal of enzyme activity and the control of the residual metabolic pathways are discussed.