Inhibitors designed for the active site of dihydroorotase

Four new compounds have been synthesized as potential inhibitors of dihydroorotase from Escherichia coli. NMR spectroscopy was used to show that 4,6-dioxo-piperidine-2(S)-carboxylic acid (3), exists in solution as a mixture of the hydrate (7), enol (8), and enolate (9) tautomeric forms. This compound was found to be a competitive inhibitor versus dihydroorotate and thio-dihydroorotate at pH values of 7-9. The K(i) of 76 microM was lowest at pH7.0 where the ketone and hydrate forms of the inhibitor 3 predominate in solution. Compound 3 was reduced to the two diastereomeric 4-hydroxy derivatives (4 and 5) and then dehydrated to yield the alkene derivative, 1,2,3,6-tetrahydro-6-oxopyridine-2(S)-carboxylic acid (6). Compounds 4-6 were competitive inhibitors versus thio-dihydroorotate at pH 8.0 with K(i) values of 3.0, 1.6, and 2.3 mM. Dihydroorotase was unable to dehydrate the 4-hydroxy derivative 4 or 5 to the alkene 6 or catalyze the reverse reaction.     

pH regulation of mitochondrial branch chain alpha-keto acid transport and oxidation in rat heart mitochondria

The kinetics of branched chain alpha-keto acid uptake and efflux were studied as a function of varied external and matrix pH. Matrix pH was determined by the distribution of 5,5'-dimethyloxazolidine-2,4-dione. When rat heart mitochondria were incubated under transport conditions at pH 7.0 with succinate as respiratory substrate, the matrix pH was significantly greater than 8.0. Matrix pH remained greater than or equal to 8.0 when the medium pH was varied from 6.3 to 8.3, and it was lowered below 8.0 by addition of 5 mM phosphate or uncoupler. No pH gradient was detectable when mitochondria were incubated in the presence of valinomycin and uncoupler. Efflux of alpha-ketoisocaproate or alpha-ketoisovalerate from rat heart mitochondria obeyed first order kinetics. Varying the external pH from 6.6 to 8.3 had no significant effect on efflux, and at an external pH of 7.0, the first order rate constant for efflux was not affected by decreasing the matrix pH. On the other hand, exchange was sensitive to changes in medium but not matrix pH. The K0.5 for external branched chain alpha-keto acid was lowered by changing the medium pH from 7.6 to 6.3. At medium pH values greater than or equal to 8.0 both K0.5 and Vmax were affected. Uptake was determined either by measuring initial rates or was calculated after measuring the first order approach to a final equilibrium value. Unlike efflux, uptake was sensitive to changes in both external and matrix pH. The rate of branched chain alpha-keto acid uptake was stimulated by decreasing the medium pH from 8.3 to 6.3 and by alkalinization of the mitochondrial matrix. The estimated external pK for proton binding was 6.9. The data indicate that the branched chain alpha-keto acid transporter is asymmetric, that is, binding sites for substrate on the inside and outside of the mitochondrial membrane are not identical. alpha-Ketoisocaproate oxidation was measured at 37 degrees C in isolated mitochondria over the pH range of 6.6 to 8.1. Changes in the rate of branched chain alpha-keto acid oxidation, particularly when ATP was added (increase delta pH), were found to parallel the pH effects observed on branched chain alpha-keto acid uptake. Therefore, transport, and by implication oxidation, can be regulated by pH changes within the physiological range. Furthermore, intracellular pH may affect the degree of compartmentation between the cytosolic and mitochondrial branched chain alpha-keto acid pools.     

Synthesis and properties of 2-acetylthiamin pyrophosphate: an enzymatic reaction intermediate

The synthesis of 2-acetylthiamin pyrophosphate (acetyl-TPP) is described. The synthesis of this compound is accomplished at 23 degrees C by the oxidation of 2-(1-hydroxyethyl)thiamin pyrophosphate using aqueous chromic acid as the oxidizing agent under conditions where Cr(III) coordination to the pyrophosphoryl moiety and hydrolysis of both the pyrophosphate and acetyl moieties were prevented. Although the chemical properties exhibited by acetyl-TPP are similar to those of the 2-acetyl-3,4-dimethylthiazolium ion examined by Lienhard [Lienhard, G.E. (1966) J. Am. Chem. Soc. 88, 5642-5649], significant differences exist because of the pyrimidine ring in acetyl-TPP. Characterization of acetyl-TPP by ultraviolet spectroscopy, 1H NMR, 13C NMR, and 31P NMR provided evidence that the compound in aqueous solution exists as an equilibrium mixture of keto, hydrate, and intramolecular carbinolamine forms. The equilibria for the hydration and carbinolamine formation reactions at pD 1.3 as determined by 1H NMR are strongly dependent on the temperature, showing an increase in the hydrate and carbinolamine forms at the expense of the keto form with decreasing temperature. The concentration of keto form also decreases with increasing pH. Acetyl-TPP is stable in aqueous acid but rapidly deacetylates at higher pH to form acetate and thiamin pyrophosphate. Trapping of the acetyl moiety in aqueous solution occurs efficiently with 1.0 M hydroxylamine at pH 5.5-6.5 to form acetohydroxamic acid and to a much smaller extent with 1.0 M 2-mercaptoethanol at pH 4.0 and 5.0 to form thio ester. Transfer of the acetyl group to 0.5 M dihydrolipoic acid at pH 5.0 and 1.0 M phosphate dianion at pH 7.0 is not observed to any significant extent in water. The kinetic and thermodynamic reactivity of acetyl-TPP with water and other nucleophiles is compatible with a hypothetical role for acyl-TPPs as enzymatic acyl-transfer intermediates.     

Enzymatic oxidation of L-homocysteine

Homocyst(e)ine, a normal metabolite, accumulates in certain inborn errors of sulfur amino acid metabolism. Since many amino acids are converted by enzymatic oxidation and by transamination to the corresponding alpha-keto acid analogs and related products, which may exert inhibitory effects on metabolism, and because the alpha-keto acid analog of homocysteine has not yet been prepared, the enzymatic oxidation of homocysteine was investigated with the aim of obtaining alpha-keto-gamma-mercaptobutyric acid. Oxidation of DL-homocysteine by L-amino acid oxidase led to formation of at least seven products that react with 2,4-dinitrophenylhydrazine; of these, five were identified: alpha-keto-gamma-mercaptobutyrate, the mono and diketo analogs of homolanthionine, and the mono and diketo analogs of homocystine. In addition, one product was tentatively identified as alpha-ketomercaptobutyric acid gamma-thiolactone. In the course of this work alpha-keto-gamma-mercaptobutyrate was found to be a substrate of lactate dehydrogenase. L-Homocysteine and its alpha-keto acid analog were shown to be substrates of glutamate dehydrogenase and kidney glutamine transaminase. DL-Homocysteine reacts readily with alpha-keto acids to form stable hemithioketals, which were found to be substrates of L- and D-amino acid oxidases. A scheme is presented which integrates some of the complexities involved in the oxidation metabolism of homocyst(e)ine. The significance of these findings is considered in relation to the toxicity of homocysteine, which accumulates in certain pathological states.     

Ability of thermophilic lactic acid bacteria to produce aroma compounds from amino acids

Although a large number of key odorants of Swiss-type cheese result from amino acid catabolism, the amino acid catabolic pathways in the bacteria present in these cheeses are not well known. In this study, we compared the in vitro abilities of Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, and Streptococcus thermophilus to produce aroma compounds from three amino acids, leucine, phenylalanine, and methionine, under mid-pH conditions of cheese ripening (pH 5.5), and we investigated the catabolic pathways used by these bacteria. In the three lactic acid bacterial species, amino acid catabolism was initiated by a transamination step, which requires the presence of an alpha-keto acid such as alpha-ketoglutarate (alpha-KG) as the amino group acceptor, and produced alpha-keto acids. Only S. thermophilus exhibited glutamate dehydrogenase activity, which produces alpha-KG from glutamate, and consequently only S. thermophilus was capable of catabolizing amino acids in the reaction medium without alpha-KG addition. In the presence of alpha-KG, lactobacilli produced much more varied aroma compounds such as acids, aldehydes, and alcohols than S. thermophilus, which mainly produced alpha-keto acids and a small amount of hydroxy acids and acids. L. helveticus mainly produced acids from phenylalanine and leucine, while L. delbrueckii subsp. lactis produced larger amounts of alcohols and/or aldehydes. Formation of aldehydes, alcohols, and acids from alpha-keto acids by L. delbrueckii subsp. lactis mainly results from the action of an alpha-keto acid decarboxylase, which produces aldehydes that are then oxidized or reduced to acids or alcohols. In contrast, the enzyme involved in the alpha-keto acid conversion to acids in L. helveticus and S. thermophilus is an alpha-keto acid dehydrogenase that produces acyl coenzymes A.     

Branched-chain alpha-keto acid catabolism via the gene products of the bkd operon in Enterococcus faecalis: a new, secreted metabolite serving as a temporary redox sink

Recently the bkd gene cluster from Enterococcus faecalis was sequenced, and it was shown that the gene products constitute a pathway for the catabolism of branched-chain alpha-keto acids. We have now investigated the regulation and physiological role of this pathway. Primer extension analysis identified the presence of a single promoter upstream of the bkd gene cluster. Furthermore, a putative catabolite-responsive element was identified in the promoter region, indicative of catabolite repression. Consistent with this was the observation that expression of the bkd gene cluster is repressed in the presence of glucose, fructose, and lactose. It is proposed that the conversion of the branched-chain alpha-keto acids to the corresponding free acids results in the formation of ATP via substrate level phosphorylation. The utilization of the alpha-keto acids resulted in a marked increase of biomass, equivalent to a net production of 0.5 mol of ATP per mol of alpha-keto acid metabolized. The pathway was active under aerobic as well as anaerobic conditions. However, under anaerobic conditions the presence of a suitable electron acceptor to regenerate NAD(+) from the NADH produced by the branched-chain alpha-keto acid dehydrogenase complex was required for complete conversion of alpha-ketoisocaproate. Interestingly, during the conversion of the branched-chain alpha-keto acids an intermediate was always detected extracellularly. With alpha-ketoisocaproic acid as the substrate this intermediate was tentatively identified as 1, 1-dihydroxy-4-methyl-2-pentanone. This reduced form of alpha-ketoisocaproic acid was found to serve as a temporary redox sink.     

D-amino acid aminotransferase of Bacillus sphaericus. Enzymologic and spectrometric properties.

D-Amino acid aminotransferase, purified to homogeneity and crystallized from Bacillus sphaericus, has a molecular weight of about 60,000 and consists of two subunits identical in molecular weight (30,000). The enzyme exhibits absorption maxima at 280, 330, and 415 nm, which are independent of the pH (5.5 to 10.0), and contains 2 mol of pyridoxal 5'-phosphate per mol of enzyme. One of the pyridoxal-5'-P, absorbing at 415 nm, is bound in an aldimine linkage to the epsilon-amino group of a lysine residue of the protein, and is released by incubation with phenylhydrazine to yield the catalytically inactive form. The inactive form, which is reactivated by addition of pyridoxal 5'phosphate, still has a 330 nm peak and contains 1 mol of pyridoxal 5'-phosphate. Therefore, this form is regarded as a semiapoenzyme. The holoenzyme shows negative circular dichroic bands at 330 and 415 nm. D-Amino acid aminotransferase catalyzes alpha transamination of various D-amino acids and alpha-keto acids. D-Alanine, D-alpha-aminobutyrate and D-glutamate, and alpha-ketoglutarate, pyruvate, and alpha-ketobutyrate are the preferred amino donors and acceptors, respectively. The enzyme activity is significantly affected by both the carbonyl and sulfhydryl reagents. The Michaelis constants are as follows: D-alanine (1.3 and 4.2 mM with alpha-ketobutyrate and alpha-ketoglutarate, respictively), alpha-ketobutyrate (14 mM withD-alanine), alpha-ketoglutarate (3.4 mM with D-alanine), pyridoxal 5'-phosphate (2.3 muM) and pyridoxamine 5'-phosphate (25 muM).     

Alpha-keto acids are novel siderophores in the genera Proteus, Providencia, and Morganella and are produced by amino acid deaminases.

Growth promotion and iron transport studies revealed that certain alpha-keto acids generated by amino acid deaminases, by enterobacteria of the Proteus-Providencia-Morganella group (of the tribe Proteeae), show significant siderophore activity. Their iron-binding properties were confirmed by the chrome azurol S assay and UV spectra. These compounds form ligand-to-metal charge transfer bands in the range of 400 to 500 nm. Additional absorption bands of the enolized ligands at 500 to 700 nm are responsible for color formation. Siderophore activity was most pronounced with alpha-keto acids possessing an aromatic or heteroaromatic side chain, like phenylpyruvic acid and indolylpyruvic acid, resulting from deamination of phenylalanine and tryptophan, respectively. In addition, alpha-keto acids possessing longer nonpolar side chains, like alpha-ketoisocaproic acid or alpha-ketoisovaleric acid and even alpha-ketoadipic acid, also showed siderophore activity which was absent or negligible with smaller alpha-keto acids or those possessing polar functional groups, like pyruvic acid, alpha-ketobutyric acid, or alpha-ketoglutaric acid. The fact that deaminase-negative enterobacteria, like Escherichia coli and Salmonella spp., could not utilize alpha-keto acids supports the view that specific iron-carboxylate transport systems have evolved in members of the tribe Proteeae and are designed to recognize ferric complexes of both alpha-hydroxy acids and alpha-keto acids, of which the latter can easily be generated by L-amino acid deaminases in an amino acid-rich medium. Exogenous siderophores, like ferric hydroxamates (ferrichromes) and ferric polycarboxylates (rhizoferrin and citrate), were also utilized by members of the tribe Proteeae.     

VALENCY RULE AND ALLEGED HOFMEISTER SERIES IN THE COLLOIDAL BEHAVIOR OF PROTEINS : I. THE ACTION OF ACIDS.

1. The action of a number of acids on four properties of gelatin (membrane potentials, osmotic pressure, swelling, and viscosity) was studied. The acids used can be divided into three groups; first, monobasic acids (HCl, HBr, HI, HNO₃, acetic, propionic, and lactic acids); second, strong dibasic acids (H₂SO₄ and sulfosalicylic acid) which dissociate as dibasic acids in the range of pH between 4.7 and 2.5; and third, weak dibasic and tribasic acids (succinic, tartaric, citric) which dissociate as monobasic acids at pH 3.0 or below and dissociate increasingly as dibasic acids, according to their strength, with pH increasing above 3.0. 2. If the influence of these acids on the four above mentioned properties of gelatin is plotted as ordinates over the pH of the gelatin solution or gelatin gel as abscissæ, it is found that all the acids have the same effect where the anion is monovalent; this is true for the seven monobasic acids at all pH and for the weak dibasic and tribasic acids at pH below 3.0. The two strong dibasic acids (the anion of which is divalent in the whole range of pH of these experiments) have a much smaller effect than the acids with monovalent anion. The weak dibasic and tribasic acids act, at pH above 3.0, like acids the anion of which is chiefly monovalent but which contain also divalent anions increasing with pH and with the strength of the acid. 3. These experiments prove that only the valency but not the other properties of the anion of an acid influences the four properties of gelatin mentioned, thus absolutely contradicting the Hofmeister anion series in this case which were due to the failure of the earlier experimenters to measure properly the pH of their protein solutions or gels and to compare the effects of acids at the same pH of the protein solution or protein gel after equilibrium was established. 4. It is shown that the validity of the valency rule and the non-validity of the Hofmeister anion series for the four properties of proteins mentioned are consequences of the fact that the influence of acids on the membrane potentials, osmotic pressure, swelling, and viscosity of gelatin is due to the Donnan equilibrium between protein solutions or gels and the surrounding aqueous solution. This equilibrium depends only on the valency but not on any other property of the anion of an acid. 5. That the valency rule is determined by the Donnan equilibrium is strikingly illustrated by the ratio of the membrane potentials for divalent and monovalent anions of acids. Loeb has shown that the Donnan equilibrium demands that this ratio should be 0.66 and the actual measurements agree with this postulate of the theory within the limits of accuracy of the measurements. 6. The valency rule can be expected to hold for only such properties of proteins as depend upon the Donnan equilibrium. Properties of proteins not depending on the Donnan equilibrium may be affected not only by the valency but also by the chemical nature of the anion of an acid.     

Transamination of L-cystathionine and related compounds by a bovine liver enzyme. Possible identification with glutamine transaminase

A transaminase which catalyses the monodeamination of L-cystathionine was purified 1100-fold with a yield of 15% from bovine liver. The monoketoderivative of cystathionine spontaneously produces the cyclic ketimine. Other sulfur-containing amino acids related to cystathionine such as cystine, lanthionine and aminoethylcysteine were also substrates for the enzyme. The relative molecular mass of the enzyme was determined to be 94 000 with a probable dimeric structure formed of identical subunits. The isoelectric point of the enzyme was at pH 5.0 and the maximal enzymatic activity was found at pH 9.0--9.2. Kinetic parameters for cystathionine and for the other sulfur amino acids as well as for some alpha-keto acids were also determined. Among the natural amino acids tested, glutamine, methionine and histidine were the best amino donors. The enzyme exhibited maximal activity toward phenylpyruvate and alpha-keto-gamma-methiolbutyrate as amino acceptors. The broad specificity of the enzyme leads us to infer that the cystathionine transaminase is very similar or identical to glutamine transaminase.     

Inhibition of glycine oxidation by pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids in rat liver mitochondria: presence of interaction between the glycine cleavage system and alpha-keto acid dehydrogenase complexes

Pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids which were transaminated products of valine, leucine, and isoleucine inhibited glycine decarboxylation by rat liver mitochondria. However, glycine synthesis (the reverse reaction of glycine decarboxylation) was stimulated by those alpha-keto acids with the concomitant decarboxylation of alpha-keto acid added in the absence of NADH. Both the decarboxylation and the synthesis of glycine by mitochondrial extract were affected similarly by alpha-ketoglutarate and branched-chain alpha-keto acids in the absence of pyridine nucleotide, but not by pyruvate. This failure of pyruvate to have an effect was due to the lack of pyruvate oxidation activity in the mitochondrial extract employed. It indicated that those alpha-keto acids exerted their effects by providing reducing equivalents to the glycine cleavage system, possibly through lipoamide dehydrogenase, a component shared by the glycine cleavage system and alpha-keto acid dehydrogenase complexes. On the decarboxylation of pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids in intact mitochondria, those alpha-keto acids inhibited one another. In similar experiments with mitochondrial extract, decarboxylations of alpha-ketoglutarate and branched-chain alpha-keto acid were inhibited by branched-chain alpha-keto acid and alpha-ketoglutarate, respectively, but not by pyruvate. NADH was unlikely to account for the inhibition. We suggest that the lipoamide dehydrogenase component is an indistinguishable constituent among alpha-keto acid dehydrogenase complexes and the glycine cleavage system in mitochondria in nature, and that lipoamide dehydrogenase-mediated transfer of reducing equivalents might regulate alpha-keto acid oxidation as well as glycine oxidation.     

Thiazolidine-2-carboxylic acid, an adduct of cysteamine and glyoxylate, as a substrate for D-amino acid oxidase

A mixture of cysteamine and glyoxylate, proposed by Hamilton et al. to form the physiological substrate of hog kidney D-amino acid oxidase (Hamilton, G. A., Buckthal, D. J., Mortensen, R. M., and Zerby, K. W. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 2625-2629), was confirmed to act as a good substrate for the pure enzyme. As proposed by those workers, it was shown that the actual substrate is thiazolidine-2-carboxylic acid, formed from cysteamine and glyoxylate with a second order rate constant of 84 min-1 M-1 at 37 degrees C, pH 7.5. Steady state kinetic analyses reveal that thiazolidine-2-carboxylic acid is a better substrate at pH 8.5 than at pH 7.5. At both pH values, the catalytic turnover number is similar to that obtained with D-proline. D-Amino acid oxidase is rapidly reduced by thiazolidine-2-carboxylic acid to form a reduced enzyme-imino acid complex, as is typical with D-amino acid oxidase substrates. The product of oxidation was shown by NMR to be delta 2-thiazoline-2-carboxylic acid. Racemic thiazolidine-2-carboxylic acid is completely oxidized by the enzyme. The directly measured rate of isomerization of L-thiazolidine-2-carboxylic acid to the D-isomer was compared to the rate of oxidation of the L-isomer by D-amino acid oxidase. Their identity over the range of temperature from 2-30 degrees C established that the apparent activity with the L-amino acid can be explained quantitatively by the rapid, prior isomerization to D-thiazolidine-2-carboxylic acid.     

Production of the apoptotic cellular mediator 4-Methylthio-2-oxobutyric acid by using an enzymatic stirred tank reactor with in situ product removal

d-Amino acid oxidase (DAAO) was used to study the oxidative deamination of racemic mixtures of d,l-methionine in its soluble and immobilized forms and thus obtain the corresponding alpha-keto acid. The soluble enzyme form was obtained from a Trigonopsis variabilis CBS 4095 extract, free of l-amino acid oxidase, and was co-immobilized with a 200-fold excess of catalase to avoid the undesirable side reaction of H2O2 with the alpha-keto acid, which would otherwise render its corresponding decarboxylated acid, the 3-methylthiopropionic acid (MTPA). With this biocatalyst, quantitative conversion (>98%) of d-methionine into the alpha-keto acid 4-methylthio-2-oxobutyric acid (MTOB) and into MTPA was achieved using 5 mg.mL(-1) of biocatalyst at pH 8.0, 25 degrees C, and pure oxygen at 3 vvm. A stirred tank reactor with in situ product removal (STR-ISPR) was developed to avoid conversion of MTOB into MTPA. The reaction medium was re-circulated through a strong anion exchange column (Amberlite IRA-400). This resulted in the complete removal of MTOB from the reaction medium. After the reaction, the reaction products were eluted sequentially with water (l-methionine), 10 mM HCl (MTPA), and 0.5 M HCl (MTOB). After elution, MTOB was crystallized to its sodium salt.     

Blood-brain barrier transport of the alpha-keto acid analogs of amino acids

A number of alpha-keto acid analogs of amino acids have been found to penetrate the blood-brain barrier (BBB). Pyruvate, alpha-ketobutyrate, alpha-ketoisocaproate, and alpha-keto-gamma-methiolbutyrate all cross the BBB by a carrier-mediated process and by simple diffusion. Under normal physiological conditions, diffusion accounts for roughly 15% or less of total transport. Aromatic alpha-keto acids, phenylpyruvate, and p-hydroxyphenylpyruvate do not penetrate the BBB, nor do they inhibit the transport of other alpha-keto acids. Evidence based primarily on inhibition studies indicates that the carrier-mediated transport of alpha-keto acids occurs via the same carrier demonstrated previously for propionate, acetoacetate, and beta-hydroxybutyrate transport, commonly referred to as the monocarboxylate carrier. As a group, the alpha-keto acid analogs of the amino acids have the highest affinity for the carrier, followed by propionate and beta-hydroxybutyrate. Starvation for 4 days induces transport of alpha-keto acids, but transport is suppressed in rats fed commercial laboratory rations and subjected to portacaval shunts. The mitochondrial pyruvate translocator inhibitor alpha-cyanocinnamate has no effect on the BBB transport of alpha-keto acids.     

Mechanism of reactions catalyzed by selenocysteine beta-lyase

The reaction mechanism of selenocystine beta-lyase has been studied and it was found that elemental selenium is released enzymatically from selenocysteine, and reduced to H2Se nonenzymatically with dithiothreitol or some other reductants that are added to prepare selenocysteine from selenocystine in the anaerobic reaction system. 1H and 13C NMR spectra of L-alanine formed in 2H2O have shown that an equimolar amount of [beta-2H1]- and [beta-2H2]alanines are produced. The deuterium isotope effect at the alpha position was observed; kH/kD = 2.4. These results indicated that the alpha hydrogen of selenocysteine was removed by a base at the active site, and was incorporated into the alpha position of alanine, a product, without exchange of a solvent deuterium. When the enzyme was incubated with L-selenocysteine in the absence of added pyridoxal 5'-phosphate, the activity decreased with prolonged incubation time. However, the activity was recovered by addition of 5'-phosphate. The spectrophotometric study showed that the inactivated enzyme was the apo form. The apoenzyme was activated by a combination of pyridoxamine 5'-phosphate and various alpha-keto acids such as alpha-ketoglutarate and pyruvate. Thus, the enzyme is inactivated through transamination between selenocysteine and the bound pyridoxal 5'-phosphate to produce pyridoxamine 5'-phosphate and a keto acid derived from selenocysteine. The pyridoxal enzyme, an active form, is regenerated by addition of alpha-keto acids. This regulatory mechanism is analogous to those of aspartate beta-decarboxylase [EC 4.1.1.12], arginine racemase [EC 5.1.1.9], and kynureninase [EC 3.7.1.3] [K. Soda and K. Tanizawa (1979) Adv. Enzymol. 49, 1].     

Fatty acid production from amino acids and alpha-keto acids by Brevibacterium linens BL2

Low concentrations of branched-chain fatty acids, such as isobutyric and isovaleric acids, develop during the ripening of hard cheeses and contribute to the beneficial flavor profile. Catabolism of amino acids, such as branched-chain amino acids, by bacteria via aminotransferase reactions and alpha-keto acids is one mechanism to generate these flavorful compounds; however, metabolism of alpha-keto acids to flavor-associated compounds is controversial. The objective of this study was to determine the ability of Brevibacterium linens BL2 to produce fatty acids from amino acids and alpha-keto acids and determine the occurrence of the likely genes in the draft genome sequence. BL2 catabolized amino acids to fatty acids only under carbohydrate starvation conditions. The primary fatty acid end products from leucine were isovaleric acid, acetic acid, and propionic acid. In contrast, logarithmic-phase cells of BL2 produced fatty acids from alpha-keto acids only. BL2 also converted alpha-keto acids to branched-chain fatty acids after carbohydrate starvation was achieved. At least 100 genes are potentially involved in five different metabolic pathways. The genome of B. linens ATCC 9174 contained these genes for production and degradation of fatty acids. These data indicate that brevibacteria have the ability to produce fatty acids from amino and alpha-keto acids and that carbon metabolism is important in regulating this event.     

Use of sulfhydryl reagents to investigate branched chain alpha-keto acid transport in mitochondria

The goal of this paper was to determine the contribution of the mitochondrial branched chain aminotransferase (BCATm) to branched chain alpha-keto acid transport within rat heart mitochondria. Isolated heart mitochondria were treated with sulfhydryl reagents of varying permeability, and the data suggest that essential cysteine residues in BCATm are accessible from the cytosolic face of the inner membrane. Treatment with 15 nmol/mg N-ethylmaleimide (NEM) inhibited initial rates of alpha-ketoisocaproate (KIC) uptake in reconstituted mitochondrial detergent extracts by 70% and in the intact organelle by 50%. KIC protected against inhibition suggesting that NEM labeled a cysteine residue that is inaccessible when substrate is bound to the enzyme. Additionally, the apparent mitochondrial equilibrium KIC concentration was decreased 50-60% after NEM labeling, and this difference could not be attributed to effects of NEM on matrix pH or KIC oxidation. In fact, NEM was a better inhibitor of KIC oxidation than rotenone. Measuring matrix aspartate and glutamate levels revealed that the effects of NEM on the steady-state KIC concentration resulted from inhibition of BCATm catalyzed transamination of KIC with matrix glutamate to form leucine. Furthermore, circular dichroism spectra of recombinant human BCATm with liposomes showed that the commercial lipids used in the reconstituted transport assay contain BCAT amino acid substrates. Thus BCATm is distinct from the branched chain alpha-keto acid carrier but may interact with the inner mitochondrial membrane, and it is necessary to inhibit or remove transaminase activity in both intact and reconstituted systems prior to quantifying transport of alpha-keto acids which are transaminase substrates.     

Role of mitochondrial transamination in branched chain amino acid metabolism

Oxidative decarboxylation and transamination of 1-14C-branched chain amino and alpha-keto acids were examined in mitochondria isolated from rat heart. Transamination was inhibited by aminooxyacetate, but not by L-cycloserine. At equimolar concentrations of alpha-ketoiso[1-14C]valerate (KIV) and isoleucine, transamination was increased by disrupting the mitochondria with detergent which suggests transport may be one factor affecting the rate of transamination. Next, the subcellular distribution of the aminotransferase(s) was determined. Branched chain aminotransferase activity was measured using two concentrations of isoleucine as amino donor and [1-14C]KIV as amino acceptor. The data show that branched chain aminotransferase activity is located exclusively in the mitochondria in rat heart. Metabolism of extramitochondrial branched chain alpha-keto acids was examined using 20 microM [1-14C]KIV and alpha-ketoiso[1-14C]caproate (KIC). There was rapid uptake and oxidation of labeled branched chain alpha-keto acid, and, regardless of the experimental condition, greater than 90% of the labeled keto acid substrate was metabolized during the 20-min incubation. When a branched chain amino acid (200 microM) or glutamate (5 mM) was present, 30-40% of the labeled keto acid was transaminated while the remainder was oxidized. Provision of an alternate amino acceptor in the form of alpha-keto-glutarate (0.5 mM) decreased transamination of the labeled KIV or KIC and increased oxidation. Metabolism of intramitochondrially generated branched chain alpha-keto acids was studied using [1-14C]leucine and [1-14C]valine. Essentially all of the labeled branched chain alpha-keto acid produced by transamination of [1-14C]leucine or [1-14C]valine with a low concentration of unlabeled branched chain alpha-keto acid (20 microM) was oxidized. Further addition of alpha-ketoglutarate resulted in a significant increase in the rate of labeled leucine or valine transamination, but again most of the labeled keto acid product was oxidized. Thus, catabolism of branched chain amino acids will be favored by a high concentration of mitochondrial alpha-ketoglutarate and low intramitochondrial glutamate.     

Possible involvement of lipoic acid in binding protein-dependent transport systems in Escherichia coli.

We describe the properties of the binding protein dependent-transport of ribose, galactose, and maltose and of the lactose permease, and the phosphoenolpyruvate-glucose phosphotransferase transport systems in a strain of Escherichia coli which is deficient in the synthesis of lipoic acid, a cofactor involved in alpha-keto acid dehydrogenation. Such a strain can grow in the absence of lipoic acid in minimal medium supplemented with acetate and succinate. Although the lactose permease and the phosphoenolypyruvate-glucose phosphotransferase are not affected by lipoic acid deprivation, the binding protein-dependent transports are reduced by 70% in conditions of lipoic acid deprivation when compared with their activity in conditions of lipoic acid supply. The remaining transport is not affected by arsenate but is inhibited by the uncoupler carbonylcyanide-m-chlorophenylhydrazone; however the lipoic acid-dependent transport is completely inhibited by arsenate and only weakly inhibited by carbonylcyanide-m-chlorophenylhydrazone. The known inhibitor of alpha-keto acid dehydrogenases, 5-methoxyindole-2-carboxylic acid, completely inhibits all binding protein-dependent transports whether in conditions of lipoic supply or deprivation; the results suggest a possible relation between binding protein-dependent transport and alpha-keto acid dehydrogenases and shed light on the inhibition of these transports by arsenicals and uncouplers.