D-glucaric acid and galactaric acid catabolism by Agrobacterium tumefaciens

Cell-free extract (crude extract) of Agrobacterium tumefaciens grown on d-glucuronate or d-glucarate converts d-glucarate and galactarate to a mixture of 2-keto-3-deoxy- and 4-deoxy-5-keto-d-glucarate. These compounds are then converted by partially purified crude extract to an intermediate tentatively identified as 2,5-diketoadipate. The same enzyme preparation further decarboxylates this intermediate to alpha-ketoglutarate semialdehyde, which is subsequently oxidized in a nicotinamide adenine dinucleotide-dependent reaction to alpha-ketoglutaric acid. Since A. tumefaciens converts d-glucuronic acid to d-glucarate, a pathway from d-glucuronate to alpha-ketoglutarate in A. tumefaciens was determined.     

The metabolism of galactarate, d-glucarate and various pentoses by species of Pseudomonas

1. When NAD⁺ was present, cell extracts of Pseudomonas (A) grown with d-glucarate or galactarate converted 1mol. of either substrate into 1mol. each of 2-oxoglutarate and carbon dioxide; 70–80% of the gas originated from C-1 of the hexarate. 2. The enzyme system that liberated carbon dioxide from galactarate was inactive in air and was stabilized by galactarate or Fe²⁺ ions; the system that acted on d-glucarate was more stable and was stimulated by Mg²⁺ ions. 3. When NAD⁺ was not added, 2-oxoglutarate semialdehyde accumulated from either substrate. This compound was isolated as its bis-2,4-dinitrophenylhydrazone, and several properties of the derivative were compared with those of the chemically synthesized material. Methods were developed for the determination of 2-oxoglutarate semialdehyde. 4. Synthetic 2-oxoglutarate semialdehyde was converted into 2-oxoglutarate by an enzyme that required NAD⁺; the reaction rate with NADP⁺ was about one-sixth of that with NAD⁺. 5. For extracts of Pseudomonas (A) grown with d-glucarate or galactarate, or for those of Pseudomonas fragi grown with l-arabinose or d-xylose, specific activities of 2-oxoglutarate semialdehyde–NAD oxidoreductase were much higher than for extracts of the organisms grown with (+)-tartrate and d-glucose respectively. 6. Extracts of Pseudomonas fragi grown with l-arabinose or d-xylose converted l-arabonate or d-xylonate into 2-oxoglutarate when NAD⁺ was added to reaction mixtures and into 2-oxoglutarate semialdehyde when NAD⁺ was omitted.     

alpha-ketoglutaric semialdehyde dehydrogenase isozymes involved in metabolic pathways of D-glucarate, D-galactarate, and hydroxy-L-proline. Molecular and metabolic convergent evolution

Azospirillum brasilense possesses an alternative pathway of l-arabinose metabolism in which alpha-ketoglutaric semialdehyde (alphaKGSA) dehydrogenase (KGSADH) is involved in the last step, the conversion of alphaKGSA to alpha-ketoglutarate. In the preceding studies, we identified a set of metabolic genes of the l-arabinose pathway including the KGSADH gene (Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 2612-2623; Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 28876-28888; Watanabe, S., Shimada, N., Tajima, K., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 33521-33536). Here, we describe that A. brasilense possesses two different KGSADH isozymes from l-arabinose-related enzyme (KGSADH-I); that is, d-glucarate/d-galactarate-inducible KGSADH-II and hydroxy-l-proline-inducible KGSADH-III. They were purified homogeneously from A. brasilense cells grown on d-galactarate or hydroxy-l-proline, respectively. When compared with KGSADH-I, amino acid sequences of KGSADH-II and KGSADH-III were significantly similar but not totally identical. Physiological characterization using recombinant enzymes revealed that KGSADH-II and KGSADH-III showed similar high substrate specificity for alphaKGSA and different coenzyme specificity; that is, NAD(+)-dependent KGSADH-II and NADP(+)-dependent KGSADH-III. In the phylogenetic tree of the aldehyde dehydrogenase (ALDH) superfamily, KGSADH-II and KGSADH-III were poorly related to the known ALDH subclasses including KGSADH-I. On the other hand, ALDH-like ycbD protein involved in d-glucarate/d-galactarate operon from Bacillus subtilis is closely related to the methylmalonyl semialdehyde dehydrogenase subclass but not A. brasilense KGSADH isozymes. To estimate the correct function, the corresponding gene was expressed, purified, and characterized. Kinetic analysis revealed the physiological role as NADP(+)-dependent KGSADH. We conclude that three different types of KGSADH appeared in the bacterial evolutional stage convergently. Furthermore, even the same pathway such as l-arabinose and d-glucarate/d-galactarate metabolism also evolved by the independent involvement of KGSADH.     

The bacterial oxidation of N-methylisonicotinate, a photolytic product of Paraquat

Two bacteria have been isolated that are capable of oxidizing N-methylisonicotinate, a photodegradation product of Paraquat (1.1'-dimethyl-4,4'-bipyridylium ion). N-Methylisonicotinate-grown cells of strain 4C1, a Gram-positive rod, oxidized 2-hydroxy-N-methylisonicotinate without lag. Cell-free extracts of these cells converted 2-hydroxyisonicotinate into 2,6-dihydroxyisonicotinate; the reaction did not require molecular oxygen. Maleamate was deamidated and maleate isomerized to fumarate by soluble enzyme systems. [(14)C]Formaldehyde was isolated as the dimedone derivative from the supernatant of a cell suspension oxidizing N-[(14)C]methylisonicotinate, and no [(14)C]-methylamine was detected. Whole cells incubated with N-methyl[carboxy-(14)C]isonicotinate released 95% of the radioactivity as (14)CO(2). The second bacterium, strain 4C2, a Gram-negative rod, did not oxidize any of the mono- or di-hydroxypyridines or their N-methyl derivatives that were available or could be synthesized; nor did cell-free extracts oxidize any of these compounds. Methylamine was oxidized by whole cells without lag; cell-free extracts converted methylamine into formaldehyde when a soluble enzyme system requiring an electron acceptor was used; formaldehyde was oxidized to formate and formate to CO(2) by enzyme systems requiring NAD(+).     

A novel alpha-ketoglutaric semialdehyde dehydrogenase: evolutionary insight into an alternative pathway of bacterial L-arabinose metabolism

Azospirillum brasilense possesses an alternative pathway of l-arabinose metabolism, which is different from the known bacterial and fungal pathways. In a previous paper (Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 2612-2623), we identified and characterized l-arabinose 1-dehydrogenase, which catalyzes the first reaction step in this pathway, and we cloned the corresponding gene. Here we focused on the fifth enzyme, alpha-ketoglutaric semialdehyde (alphaKGSA) dehydrogenase, catalyzing the conversion of alphaKGSA to alpha-ketoglutarate. alphaKGSA dehydrogenase was purified tentatively as a NAD(+)-preferring aldehyde dehydrogenase (ALDH) with high activity for glutaraldehyde. The gene encoding this enzyme was cloned and shown to be located on the genome of A. brasilense separately from a gene cluster containing the l-arabinose 1-dehydrogenase gene, in contrast with Burkholderia thailandensis in which both genes are located in the same gene cluster. Higher catalytic efficiency of ALDH was found with alphaKGSA and succinic semialdehyde among the tested aldehyde substrates. In zymogram staining analysis with the cell-free extract, a single active band was found at the same position as the purified enzyme. Furthermore, a disruptant of the gene did not grow on l-arabinose. These results indicated that this ALDH gene was the only gene of the NAD(+)-preferring alphaKGSA dehydrogenase in A. brasilense. In the phylogenetic tree of the ALDH family, alphaKGSA dehydrogenase from A. brasilense falls into the succinic semialdehyde dehydrogenase (SSALDH) subfamily. Several putative alphaKGSA dehydrogenases from other bacteria belong to a different ALDH subfamily from SSALDH, suggesting strongly that their substrate specificities for alphaKGSA are acquired independently during the evolutionary stage. This is the first evidence of unique "convergent evolution" in the ALDH family.     

Alpha-ketoglutarate dehydrogenase complex of Acetobacter xylinum. Purification and regulatory properties.

The alpha-ketoglutarate dehydrogenase complex of Acetobacter xylinum was purified to homogeneity. It consists of three main polypeptide chains with a total molecular weight of about 2.4 X 10(6). It catalyzes the overall Mg2+ and thiamin pyrophosphate-dependent, NAD+- and CoA-linked oxidative decarboxylation of alpha-ketoglutarate, as well as the partial reactions characteristic of the three enzyme components described for the complex from other sources. Initial velocity studies revealed marked positive cooperativity for the substrate alpha-ketoglutarate (Hill coefficient (nH) = 2.0; concentration of ligand at half-maximum effect (S0.5) = 8 mM). The sigmoidal [alpha-ketoglutarate]-velocity relationship became hyperbolic upon addition of AMP or 3-acetylpyridine adenine dinucleotide (AcPyAD) or in the presence of high concentrations of NAD. S0.5 (alpha-ketoglutarate) decreased to 1 mM, but Vmax was unchanged. Saturation curves for NAD and AMP are sigmoidal (nH = 2) at low alpha-ketoglutarate concentrations and become hyperbolic at high alpha-ketoglutarate concentrations. As judged by S0.5, the relative efficiency of the allosteric effectors is AcPyAD greater than AMP greater than alpha-ketoglutarate- greater than NAD+. Half-maximal changes in nH, S0.5, and activation by AMP occur at a pH significantly different from that of half-maximal activity. A model for the allosteric behavior of the complex is proposed in which the first enzyme component of the complex (E1) is the site for the allosteric interactions and AMP is the primary positive modifier, whereas NAD and AcPyAD act as AMP analogues. The overall reaction is competitively inhibited by NADH with respect to NAD (K1 = 20 micronM) and by succinyl-CoA with respect of CoA (K1 = 3 micronM). The properties of the alpha-ketoglutarate dehydrogenase complex of A. xylinum appear to provide for appropriate partitioning of alpha-ketoglutarate carbon between competing pathways in response to the energy state of the cells.     

Isolation and characterization of a hydroxyacid-oxoacid transhydrogenase from rat kidney mitochondria

A transhydrogenase that catalyzes the oxoacid-dependent oxidation of specific hydroxyacids has been found in rat kidney, liver, and brain. The hydroxyacids that have been found to be substrates for this enzyme are gamma-hydroxybutyrate, D-alpha-hydroxyglutarate, and L-beta-hydroxybutyrate. The oxoacids that are the best substrates for this enzyme are alpha-ketoglutarate and succinic semialdehyde; alpha-ketoadipate and oxalacetate are also substrates. This enzyme is located in the mitochondrial fraction of the cell and is not dependent on added NAD+ or NADP+.     

Studies on the Physiology of Bacillus fastidiosus

Bacillus fastidiosus was grown in a minimal medium that contained uric acid or allantoin, aerated by vigorous stirring. A constant, optimum pH of 7.4 was maintained by controlled addition of sulfuric acid. Washed cells converted both urate and allantoin into carbon dioxide and ammonia, simultaneously assimilating part of the available carbon and nitrogen. Urate oxidase (formerly called uricase) was present in extracts from urate-grown but not allantoin-grown cells. The formation of urate oxidase was apparently induced by urate. Urea was detected as an intermediate in some but not all of these experiments. However, the high urease activity observed in cell-free extracts may have prevented accumulation of urea in many of the experiments. The presence of glyoxylate carboligase and tartronic semialdehyde reductase activities indicates that the glycerate pathway may be involved in urate and allantoin catabolism in this organism.     

Pathway for Biodegradation of p-Nitrophenol in a Moraxella sp

A Moraxella strain grew on p-nitrophenol with stoichiometric release of nitrite. During induction of the enzymes for growth on p-nitrophenol, traces of hydroquinone accumulated in the medium. In the presence of 2,2′-dipyridyl, p-nitrophenol was converted stoichiometrically to hydroquinone. Particulate enzymes catalyzed the conversion of p-nitrophenol to hydroquinone in the presence of NADPH and oxygen. Soluble enzymes catalyzed the conversion of hydroquinone to γ-hydroxymuconic semialdehyde, which was identified by high-performance liquid chromatography (HPLC)-mass spectroscopy. Upon addition of catalytic amounts of NAD⁺, γ-hydroxymuconic semialdehyde was converted to β-ketoadipic acid. In the presence of pyruvate and lactic dehydrogenase, substrate amounts of NAD were required and γ-hydroxymuconic semialdehyde was converted to maleylacetic acid, which was identified by HPLC-mass spectroscopy. Similar results were obtained when the reaction was carried out in the presence of potassium ferricyanide. Extracts prepared from p-nitrophenol-growth cells also contained an enzyme that catalyzed the oxidation of 1,2,4-benzenetriol to maleylacetic acid. The enzyme responsible for the oxidation of 1,2,4-benzenetriol was separated from the enzyme responsible for hydroquinone oxidation by DEAE-cellulose chromatography. The results indicate that the pathway for biodegradation of p-nitrophenol involves the initial removal of the nitro group as nitrite and formation of hydroquinone. 1,4-Benzoquinone, a likely intermediate in the initial reaction, was not detected. Hydroquinone is converted to β-ketoadipic acid via γ-hydroxymuconic semialdehyde and maleylacetic acid.     

Insensitivity of Homocitrate Synthase in Extracts of Penicillium chyrosogenum to Feedback Inhibition by Lysine

We previously reported that lysine inhibits in vivo homocitrate synthesis in the lysine bradytroph, Penicillium chrysogenum L(1), and that such feedback inhibition could explain the known lysine inhibition of penicillin formation. In the present study, it was found that dialyzed cell-free extracts of mutant L(1) converted [1-(14)C]acetate to homocitrate. This homocitrate synthase activity was extremely labile but could be stabilized by high salt concentrations. The pH optimum of the reaction was 6.9, and the K(m) was 5.5 mM with respect to alpha-ketoglutarate. The reaction was also dependent upon the presence of Mg(2+), adenosine 5'-triphosphate, and coenzyme A. Surprisingly, the activity in these crude extracts was not inhibited by lysine. Benzylpenicillin at a high concentration (20 mM) partially inhibited the enzyme, an effect that was enhanced by lysine. Casein hydrolysate also partially inhibited the enzyme.     

Degradation of p-toluenesulphonic acid via sidechain oxidation, desulphonation and meta ring cleavage in Pseudomonas (Comamonas) testosteroni T-2

Pseudomonas (Comamonas) testosteroni T-2 completely converted p-toluenesulphonic acid (TS) or p-sulphobenzoic acid (PSB) to cell material, CO2 and sulphate, with growth yields of about 5 g protein (mol C)-1. PSB and sulphite were excreted as transient intermediates during growth in TS-salts medium. All reactions of a catabolic pathway involving sidechain oxidation and cleavage of the sulphonate moiety as sulphite were measurable in the soluble portion of cell extracts. Degradation of TS and PSB was inducible and apparently involved at least two regulons. TS was converted to p-sulphobenzyl alcohol in a reaction requiring NAD(P)H and 1 mol O2 (mol TS)-1. This alcohol was in an equilibrium (in the presence of NAD+) with p-sulphobenzaldehyde, which was converted to PSB in an NAD(P)+-dependent reaction. PSB was desulphonated to protocatechuic acid in a reaction requiring NAD(P)H and 1 mol O2 (mol PSB)-1. Experiments with 18 O2 confirmed involvement of a dioxygenase, because both atoms of this molecular oxygen were recovered in protocatechuate. Protocatechuate was converted to 2-hydroxy-4-carboxymuconate semialdehyde by a 4.5-dioxygenase.     

Metabolism of beta-methylaspartate by a pseudomonad

A bacterium was isolated from soil which utilizes threo-beta-methyl-l-aspartate, certain other amino acids, and a variety of organic substances as single energy sources. It is, or closely resembles, Pseudomonas putida biotype B. The ability of this organism to rapidly decompose such amino acids is dependent on inducible enzyme systems. Dialyzed cell-free extracts of this bacterium metabolize beta-methylaspartate only when catalytic amounts of alpha-ketoglutarate, or pyruvate, and pyridoxal phosphate are also present. The main products formed from beta-methylaspartate under these conditions are alpha-aminobutyrate, carbon dioxide, and alpha-ketobutyrate. When l-aspartate is substituted for beta-methylaspartate in this system, it is converted mainly to alanine and carbon dioxide. beta-Methyloxalacetate is decarboxylated, and the resulting alpha-ketobutyrate is converted enzymatically in the presence of glutamate to alpha-aminobutyrate which accumulates. The added keto acids are converted, in part, to the corresponding amino acids probably by transamination. The data indicate that beta-methylaspartate is converted to alpha-aminobutyrate, and aspartate to alanine, by a circuitous transamination-beta-decarboxylation-transamination sequence rather than by a direct beta-decarboxylation.     

Catabolism of alpha-ketoglutarate by a sucA mutant of Bradyrhizobium japonicum: evidence for an alternative tricarboxylic acid cycle

A complete tricarboxylic acid (TCA) cycle is generally considered necessary for energy production from the dicarboxylic acid substrates malate, succinate, and fumarate. However, a Bradyrhizobium japonicum sucA mutant that is missing alpha-ketoglutarate dehydrogenase is able to grow on malate as its sole source of carbon. This mutant also fixes nitrogen in symbiosis with soybean, where dicarboxylic acids are its principal carbon substrate. Using a flow chamber system to make direct measurements of oxygen consumption and ammonium excretion, we confirmed that bacteroids formed by the sucA mutant displayed wild-type rates of respiration and nitrogen fixation. Despite the absence of alpha-ketoglutarate dehydrogenase activity, whole cells of the mutant were able to decarboxylate alpha-[U-(14)C]ketoglutarate and [U-(14)C]glutamate at rates similar to those of wild-type B. japonicum, indicating that there was an alternative route for alpha-ketoglutarate catabolism. Because cell extracts from B. japonicum decarboxylated [U-(14)C]glutamate very slowly, the gamma-aminobutyrate shunt is unlikely to be the pathway responsible for alpha-ketoglutarate catabolism in the mutant. In contrast, cell extracts from both the wild type and mutant showed a coenzyme A (CoA)-independent alpha-ketoglutarate decarboxylation activity. This activity was independent of pyridine nucleotides and was stimulated by thiamine PP(i). Thin-layer chromatography showed that the product of alpha-ketoglutarate decarboxylation was succinic semialdehyde. The CoA-independent alpha-ketoglutarate decarboxylase, along with succinate semialdehyde dehydrogenase, may form an alternative pathway for alpha-ketoglutarate catabolism, and this pathway may enhance TCA cycle function during symbiotic nitrogen fixation.     

Degradation of nitrobenzene by a Pseudomonas pseudoalcaligenes.

A Pseudomonas pseudoalcaligenes able to use nitrobenzene as the sole source of carbon, nitrogen, and energy was isolated from soil and groundwater contaminated with nitrobenzene. The range of aromatic substrates able to support growth was limited to nitrobenzene, hydroxylaminobenzene, and 2-aminophenol. Washed suspensions of nitrobenzene-grown cells removed nitrobenzene from culture fluids with the concomitant release of ammonia. Nitrobenzene, nitrosobenzene, hydroxylaminobenzene, and 2-aminophenol stimulated oxygen uptake in resting cells and in extracts of nitrobenzene-grown cells. Under aerobic and anaerobic conditions, crude extracts converted nitrobenzene to 2-aminophenol with oxidation of 2 mol of NADPH. Ring cleavage, which required ferrous iron, produced a transient yellow product with a maximum A380. In the presence of NAD, the product disappeared and NADH was produced. In the absence of NAD, the ring fission product was spontaneously converted to picolinic acid, which was not further metabolized. These results indicate that the catabolic pathway involves the reduction of nitrobenzene to nitrosobenzene and then to hydroxylaminobenzene; each of these steps requires 1 mol of NADPH. An enzyme-mediated Bamberger-like rearrangement converts hydroxylaminobenzene to 2-aminophenol, which then undergoes meta ring cleavage to 2-aminomuconic semialdehyde. The mechanism for release of ammonia and subsequent metabolism are under investigation.     

Biosynthesis of Protein Amino Acids in Plant Tissue Culture. III. Studies on the Biosynthesis of Arginine

Evidence from isotope competition studies and enzymic studies indicates that n-acetyl glutamic semialdehyde, alpha-n-acetyl-l-ornithine, l-ornithine and l-citrulline are intermediates between glucose and arginine in cells of Paul's Scarlet Rose. Evidence for the presence of alpha-n-acetyl-ornithine aminotransferase (E. C. 2.6.1.11) in cell-free extracts was obtained.     

Biosynthesis of o-succinylbenzoic acid. II: Properties of o-succinylbenzoic acid synthase, an enzyme involved in vitamin K2 biosynthesis

The enzyme system (OSB2 synthase) catalyzing the synthesis of o-succinylbenzoic acid from isochorismic acid and alpha-ketoglutaric acid in the presence of thiamine pyrophosphate was isolated from Escherichia coli AN 154 and characterized. The purification factor of the enzyme did not increase during column chromatography on Sephadex G-200 or chromatofocusing, suggesting that the OSB synthase is labile. Chromatography on DEAE-Sephadex A-50 or A-25 showed that an enzyme activity separated from fractions containing OSB synthase that decarboxylates alpha-ketoglutarate. This activity is provisionally referred to as the decarboxylating "subunit" or decarboxylating activity of OSB synthase. Both the "subunit" and the holoenzyme were characterized with respect to pH optimum, temperature optimum, and KM values. The OSB synthase loses all activity during treatment with EDTA and activity is most efficiently restored with Mn2+. The activity of the decarboxylating subunit did not depend on Mn2+. When the decarboxylating fraction was incubated with alpha-ketoglutarate and thiamine pyrophosphate, succinic semialdehyde could be isolated as its hydrazone. After treatment of the incubation mixture with phosphorylase a compound was isolated which is most likely the thiamine adduct of succinic semialdehyde.     

Studies of the catabolic pathway of degradation of nitrobenzene by Pseudomonas pseudoalcaligenes JS45: removal of the amino group from 2-aminomuconic semialdehyde.

Pseudomonas pseudoalcaligenes JS45 utilizes nitrobenzene as the sole source of nitrogen, carbon, and energy. Previous studies have shown that degradation of nitrobenzene involves the reduction of nitrobenzene to nitrosobenzene and hydroxylaminobenzene, followed by rearrangement to 2-aminophenol, which then undergoes meta ring cleavage to 2-aminomuconic semialdehyde. In the present paper, we report the enzymatic reactions responsible for the release of ammonia after ring cleavage. 2-Aminomuconic semialdehyde was oxidized to 2-aminomuconate in the presence of NAD by enzymes in crude extracts. 2-Aminomuconate was subsequently deaminated stoichiometrically to 4-oxalocrotonic acid. No cofactors are required for the deamination. Two enzymes, 2-aminomuconic semialdehyde dehydrogenase and a novel 2-aminomuconate deaminase, distinguished by partial purification of the crude extracts, catalyzed the two reactions. 4-Oxalocrotonic acid was further degraded to pyruvate and acetaldehyde. The key enzyme, 2-aminomuconate deaminase, catalyzed the hydrolytic deamination that released ammonia, which served as the nitrogen source for growth of the organism.     

Isocitrate Dehydrogenase and Glutamate Synthesis in Acetobacter suboxydans

Acetobacter suboxydans is an obligate aerobe for which an operative tricarboxylic acid cycle has not been demonstrated. Glutamate synthesis has been reported to occur by mechanisms other than those utilizing isocitrate dehydrogenase, a tricarboxylic acid cycle enzyme not previously detected in this organism. We have recovered alpha-ketoglutarate and glutamate from a system containing citrate, nicotinamide adenine dinucleotide (NAD), a divalent cation, pyridoxal phosphate, an amino donor, and dialyzed, cell-free extract. Aconitase activity was readily detected in these extracts, but isocitrate dehydrogenase activity, measured by NAD reduction, was masked by a cyanide-resistant, particulate, reduced NAD oxidase. Isocitrate dehydrogenase activity could be demonstrated after centrifuging the extracts at 150,000 x g for 3 hr and treating the supernatant fluid with 2-heptyl-4-hydroxyquinoline N-oxide. It is concluded that A. suboxydans can utilize the conventional tricarboxylic acid cycle enzymes to convert citrate to alpha-ketoglutarate which can then undergo a transamination to glutamate.     

Presence and regulation of the alpha-ketoglutarate dehydrogenase multienzyme complex in the filamentous fungus Aspergillus niger.

alpha-Ketoglutarate dehydrogenase has been demonstrated for the first time in cell extracts from the filamentous fungus Aspergillus niger. A minimum protein concentration of 5 mg/ml is necessary for detecting enzyme activity, but a maximum of ca. 0.060 mumol/min per mg of protein is observed only when the protein concentration is above 9 mg/ml. alpha-Ketoglutarate can partly stabilize the enzyme against dilution in the assay system. Neither bovine serum albumin nor a variety of substrates or effectors of the enzyme could stabilize the enzyme against inactivation by dilution. A kinetic analysis of the enzyme revealed Michaelis-Menten kinetics with respect to alpha-ketoglutarate, coenzyme A, and NAD. Thiamine PPi was required for maximal activity. NADH, oxaloacetate, succinate, and cis-aconitate were found to inhibit the enzyme; AMP was without effect. Monovalent cations including NH4+ were inhibitory at high concentrations (greater than 20 mM). The highest enzyme activity was found in rapidly growing mycelia (glucose-NH4+ or glucose-peptone medium). We discuss the possibility that citric acid accumulation is caused by oxaloacetate and NADH inhibition of the alpha-ketoglutarate dehydrogenase of A. niger.     

Brain succinic semialdehyde dehydrogenase. Reactions of sulfhydryl residues connected with catalytic activity.

Incubation of an NAD+-dependent succinic semialdehyde dehydrogenase from bovine brain with 4-dimethylaminoazobenzene-4-iodoacetamide (DABIA) resulted in a time-dependent loss of enzymatic activity. This inactivation followed pseudo first-order kinetics with a second-order rate constant of 168 m(-1).min(-1). The spectrum of DABIA-labeled enzyme showed a characteristic peak of the DABIA alkylated sulfhydryl group chromophore at 436 nm, which was absent from the spectrum of the native enzyme. A linear relationship was observed between DABIA binding and the loss of enzyme activity, which extrapolates to a stoichiometry of 8.0 mol DABIA derivatives per mol enzyme tetramer. This inactivation was prevented by preincubating the enzyme with substrate, succinic semialdehyde, but not by preincubating with coenzyme NAD+. After tryptic digestion of the enzyme modified with DABIA, two peptides absorbing at 436 nm were isolated by reverse-phase HPLC. The amino acid sequences of the DABIA-labeled peptides were VCSNQFLVQR and EVGEAICTDPLVSK, respectively. These sites are identical to the putative active site sequences of other brain succinic semialdehyde dehydrogenases. These results suggest that the catalytic function of succinic semialdehyde dehydrogenase is inhibited by the specific binding of DABIA to a cysteine residue at or near its active site.