Catabolism of threonine in mammals by coupling of l-threonine 3-dehydrogenase with 2-amino-3-oxobutyrate-CoA ligase

There is doubt about the l-threonine 3-dehydrogenase (EC 1.1.1.103) and threonine aldolase (EC 2.1.2.1) catabolic pathways of l-threonine in mammals which are believed to produce aminoacetone and glycine plus acetaldehyde, respectively. l-Threonine 3-dehydrogenase in disrupted guinea-pig liver mitochondria was investigated in a reaction mixture containing l-threonine without and with CoA and oxaloacetate; l-[U-¹⁴C]threonine was included in four similar experiments for autoradiograms. Threonine aldolase was examined in similar mitochondria from liver and kidney. CoA reduced the aminoacetone formed from l-threonine to 10–14% and CoA plus oxaloacetate produced citrate (from CoASAc) in approximately equal amounts to the decrease in aminoacetone. Autoradiograms confirmed the decrease in aminoacetone with the simultaneous appearance of citrate and glycine. No evidence was obtained that threonine aldolase catabolised l-threonine at the concentration used to assay the dehydrogenase. It is concluded that 2-amino-3-oxobutyrate (precursor of aminoacetone), which is produced from l-threonine by l-threonine 3-dehydrogenase, undergoes CoA-dependent cleavage to glycine and CoASAc by 2-amino-3-oxobutyrate-CoA ligase. The results suggest that the coupling of these enzymes provides a new pathway for the catabolism of threonine in mammals.     

Formation of glycine and aminoacetone from l-threonine by rat liver mitochondria

Threonine is a precursor of glycine in the rat, but the metabolic pathway involved is unclear. To elucidate this pathway, the biosynthesis of glycine, and of aminoacetone, from l-threonine were studied in rat liver mitochondrial preparations of differing integrities. In the absence of added cofactors, intact mitochondria formed glycine and aminoacetone in approximately equal amounts from 20 mM l-threonine, but exogenous NAD⁺ decreased and CoA increased the ratio of glycine to aminoacetone formed. In intact and freeze-thawed mitochondria, the ratio of glycine to aminoacetone formed was markedly sensitive to the concentration of l-threonine, glycine being the major product at low l-threonine concentrations. Disruption of mitochondrial integrity by sonication (1 min) decreased the ratio of glycine to aminoacetone formed, and in 20 000 × g supernatant fractions from sonicated (3 min) mitochondria, aminoacetone was the major product. The main non-nitogenous tow-carbon compound detected when intact mitochondria catabolized l-threonine to glycine was acetate, which was probably derived from deacylation of acetyl-CoA. These results suggest that glycine formation from l-threonine in rat liver mitochondria occured primarily by the coupled activities of threonine dehydrogenase and 2-amino-3-oxobutyrate CoA-ligase, the extent of coupling between the enzymes being dependent upon a close physical relationship and upon the flux through the dehydrogenase reaction. In vivo glycine synthesis would predominate, and aminoacetone would be a minor product.     

Utilization of l-threonine by a species of Arthrobacter. A novel catabolic role for `aminoacetone synthase''

1. A species of Arthrobacter (designated Arthrobacter 9759) was isolated from soil by its ability to grow aerobically on l-threonine as sole source of carbon atoms, nitrogen atoms and energy; the organism also grew well on other sources of carbon atoms including glycine, but no growth was obtainable on aminoacetone or dl-1-aminopropan-2-ol. 2. During growth on threonine, (14)C from l-[U-(14)C]threonine was rapidly incorporated into glycine and citrate, and thereafter into serine, alanine, aspartate and glutamate. 3. With extracts of threonine-grown cells supplied with l-[U-(14)C]threonine, evidence was obtained of the NAD and CoA-dependent catabolism of l-threonine to produce acetyl-CoA plus glycine. Short-term incorporation studies in which [2-(14)C]acetate and [2-(14)C]glycine were supplied (a) to cultures growing on threonine, and (b) to extracts of threonine-grown cells, showed that the acetyl-CoA was metabolized via the tricarboxylic acid cycle and glyoxylate cycle whereas the glycine was converted into pyruvate via the folate-dependent ;serine pathway'. 4. The threonine-grown organism contained ;biosynthetic' threonine dehydratase and a potent NAD-linked l-threonine dehydrogenase but possessed no l-threonine aldolase activity. 5. Evidence was obtained that the acetyl-CoA and glycine produced from l-threonine had their immediate origin in the alpha-amino-beta-oxobutyrate formed by the threonine dehydrogenase; the CoA-dependent cleavage of this compound was catalysed by an alpha-amino-beta-oxobutyrate CoA-ligase, which was identified with ;aminoacetone synthase'. A continuous spectrophotometric assay of this enzyme was developed, and it was found to be inducibly synthesized only during growth on threonine and not during growth on acetate plus glycine. 6. By using a reconstituted mixture of separately purified l-threonine dehydrogenase and alpha-amino-beta-oxobutyrate CoA-ligase (i.e. ;aminoacetone synthase'), l-[U-(14)C]threonine was broken down to [(14)C]glycine plus [(14)C]acetyl-CoA (trapped as [(14)C]citrate). 7. There was no evidence of aminoacetone metabolism by Arthrobacter 9759 even though a small amount of this amino ketone appeared in the culture medium during growth on threonine.     

Formation of glycine and aminoacetone from L-threonine by rat liver mitochondria

Threonine is a precursor of glycine in the rat, but the metabolic pathway involved is unclear. To elucidate this pathway, the biosynthesis of glycine, and of aminoacetone, from L-threonine were studied in rat liver mitochondrial preparations of differing integrities. In the absence of added cofactors, intact mitochondria formed glycine and aminoacetone in approximately equal amounts from 20 mM L-threonine, but exogenous NAD+ decreased and CoA increased the ratio of glycine to aminoacetone formed. In intact and freeze-thawed mitochondria, the ratio of glycine to aminoacetone formed was markedly sensitive to the concentration of L-threonine, glycine being the major product at low L-threonine concentrations. Disruption of mitochondrial integrity by sonication (1 min) decreased the ratio of glycine to aminoacetone formed, and in 20000 X g supernatant fractions from sonicated (3 min) mitochondria, aminoacetone was the major product. The main non-nitogenous two-carbon compound detected when intact mitochondria catabolized L-threonine to glycine was acetate, which was probably derived from deacylation of acetyl-CoA. These results suggest that glycine formation from L-threonine in rat liver mitochondria occurred primarily by the coupled activities of threonine dehydrogenase and 2-amino-3-oxobutyrate CoA-ligase, the extent of coupling between the enzymes being dependent upon a close physical relationship and upon the flux through the dehydrogenase reaction. In vivo glycine synthesis would predominate, and aminoacetone would be a minor product.     

Serine hydroxymethyltransferase and threonine aldolase: are they identical?

Serine hydroxymethyltransferase, a pyridoxal phosphate-dependent enzyme, catalyses the interconversion of serine and glycine, both of which are major sources of one-carbon units necessary for the synthesis of purine, thymidylate, methionine, and so on. Threonine aldolase catalyzes the pyridoxal phosphate-dependent, reversible reaction between threonine and acetaldehyde plus glycine. No extensive studies have been carried out on threonine aldolase in animal tissues, and it has long been believed that serine hydroxymethyltransferase and threonine aldolase are the same, i.e. one entity. This is based on the finding that rabbit liver serine hydroxymethyltransferase possesses some threonine aldolase activity. Recently, however, many kinds of threonine aldolase and corresponding genes were isolated from micro-organisms, and these enzymes were shown to be distinct from serine hydroxymethyltransferase. The experiments with isolated hepatocytes and cell-free extracts from various animals revealed that threonine is degraded mainly through the pathway initiated by threonine 3-dehydrogenase, and there is little or no contribution by threonine aldolase. Thus, although serine hydroxymethyltransferase from some mammalian livers exhibits a low threonine aldolase activity, the two enzymes are distinct from each other and mammals lack the "genuine" threonine aldolase.     

Purification and properties of aminoacetone synthetase from beef liver mitochondria

Aminoacetone synthetase from beef liver mitochondria was purified to homogeneity and shown to be a member of the pyridoxal 5'-phosphate-dependent family of enzymes. This enzyme catalyzes the condensation of glycine and acetyl-CoA to produce CO2, CoA, and the stable product aminoacetone. Bovine aminoacetone synthetase is a dimer (Mr 56,000) of identical subunits and contains 2 mol of pyridoxal phosphate/mol of dimer. The holoenzyme was resolved by dialysis against cysteine and has a pI of 5.2. The holoenzyme shows an absorption maximum at 428 nm which undergoes a shift to 335 nm when reduced with sodium borohydride. The Km values of glycine and acetyl-CoA were 22 mM and 53 microM, respectively. Initial velocity studies indicate that the condensation reaction proceeds by an ordered mechanism. With the exception of aminomalonate, bovine aminoacetone synthetase acts specifically on glycine and acetyl-CoA. Coupled reactions of purified bovine aminoacetone synthetase and porcine L-threonine dehydrogenase demonstrated the interconversion of threonine and glycine.     

Threonine formation via the coupled activity of 2-amino-3-ketobutyrate coenzyme A lyase and threonine dehydrogenase.

The enzymes L-threonine dehydrogenase and 2-amino-3-ketobutyrate coenzyme A (CoA) lyase are known to catalyze the net conversion of L-threonine plus NAD+ plus CoA to NADH plus glycine plus acetyl-CoA. When homogeneous preparations of these two enzymes from Escherichia coli were incubated together for 40 min at 25 degrees C with glycine, acetyl-CoA, and NADH, a 36% decrease in the level of glycine (with concomitant NADH oxidation) was matched by formation of an equivalent amount of threonine, indicating that this coupled sequence of enzyme-catalyzed reactions is reversible in vitro. Several experimental factors that affect the efficiency of this conversion in vitro were examined. A constructed strain of E. coli, MD901 (glyA thrB/C tdh), was unable to grow unless both glycine and threonine were added to defined rich medium. Introduction of the plasmid pDR121 (tdh+kbl+) into this strain enabled the cells to grow in the presence of either added glycine or threonine, indicating that interconversion of these two amino acids occurred. Threonine that was isolated from the total pool of cellular protein of MD901/pDR121 had the same specific radioactivity as the [14C]glycine added to the medium, establishing that threonine was formed exclusively from glycine in this strain. Comparative growth rate studies with several strains of E. coli containing plasmid pDR121, together with the finding that kcat values of pure E. coli 2-amino-3-ketobutyrate CoA lyase favor the cleavage of 2-amino-3-ketobutyrate over its formation by a factor of 50, indicate that the biosynthesis of threonine is less efficient than glycine formation via the coupled threonine dehydrogenase-2-amino-3-ketobutyrate lyase reactions.     

Interaction between L-threonine dehydrogenase and aminoacetone synthetase and mechanism of aminoacetone production

A mixture of threonine dehydrogenase and aminoacetone synthetase will catalyze the conversion of L-threonine to glycine. The overall reaction likely involves the conversion of L-threonine, NAD+, and CoA to glycine, NADH, and acetyl-CoA. Physical separation of L-threonine dehydrogenase from aminoacetone synthetase results in the formation of aminoacetone and CO2 from their substrates. A physical interaction between threonine dehydrogenase and aminoacetone synthetase has been demonstrated by gel permeation chromatography and fluorescence polarization. Polarization of fluorescence measurements of threonine dehydrogenase and aminoacetone synthetase labeled with fluorescein isothiocyanate indicated the formation of a soluble active complex, with an apparent dissociation constant (Kd) of 5-10 nM and an apparent stoichiometry of 2 aminoacetone synthetase dimers/1 threonine dehydrogenase tetramer. Chemical experiments have identified aminoacetone as the enzymatic product of L-threonine dehydrogenase acting on L-threonine. These experiments involved trapping pyrrole derivatives, [3H]NaBH4 reduction, and coupling with plasma amine oxidase. Kinetic experiments also showed NADH, CO2, and aminoacetone to inhibit threonine dehydrogenase in a manner consistent with an ordered Bi-Ter kinetic mechanism. NAD+ is the lead substrate followed by threonine, and the products are released in the order: CO2, aminoacetone, and NADH.     

Threonine degradation by Serratia marcescens.

The wild strain of Serratia marcescens rapidly degraded threonine and formed aminoacetone in a medium containing glucose and urea. Extracts of this strain showed high threonine dehydrogenase and "biosynthetic" threonine deaminase activities, but no threonine aldolase activity. Threonine dehydrogenase-deficient strain Mu-910 was selected among mutants unable to grow on threonine as the carbon source. This strain did not form aminoacetone from threonine, but it slowly degraded threonine. Strain D-60, deficient in both threonine dehydrogenase and threonine deaminase, was derived from strain Mu-910 and barely degraded threonine. A glycine-requiring strain derived from the wild strain grew in minimal medium containing threonine as the glycine source, whereas a glycine-requiring strain derived from strain Mu-910 did not grow. This indicates that threonine dehydrogenase participates in glycine formation from threonine (via alpha-amino-beta-ketobutyrate) as well as in threonine degradation to aminoacetone.     

Bacterial catabolism of threonine. Threonine degradation initiated by L-threonine-NAD+ oxidoreductase.

1. Isolates representing seven bacterial genera capable of growth on L-threonine medium, and possessing high L-threonine 3-dehydrogenase activity, were examined to elucidate the catabolic route. 2. The results of growth, manometric and enzymic experiments indicated the catabolism of L-threonine by cleavage to acetyl-CoA plus glycine, the glycine being further metabolized via L-serine to pyruvate, in all cases. No evidence was obtained of a role for aminoacetone in threonine catabolism or for the metabolism of glycine by the glycerate pathway. 3. The properties of a number of key enzymes in L-threonine catabolism were investigated. The inducibly formed L-threonine 3-dehydrogenase, purified from Corynebacterium sp. B6 to a specific activity of about 30-35 mumol of product formed/min per mg of protein, exhibited a sigmoid kinetic response to substrate concentration. The half-saturating concentration of substrate, [S]0.5, was 20mM and the Hill constant (h) was 1.50. The Km for NAD+ was 0.8mM. The properties of the enzyme were studied in cell-free extracts of other bacteria. 4. New assays for 2-amino-3-oxobutyrate-CoA ligase were devised. The Km for CoA was determined for the first time and found to be 0.14mM at pH8, for the enzyme from Corynebacterium sp. B6. Evidence was obtained for the efficient linkage of the dehydrogenase and ligase enzymes. Cell-free extracts all possessed high activities of the inducibly formed ligase. 5. L-Serine hydroxymethyltransferase was formed constitutively by all isolates, whereas formation of the 'glycine-cleavage system' was generally induced by growth on L-threonine or glycine. The coenzyme requirements of both enzymes were established, and their linked activity in the production of L-serine from glycine was demonstrated by using extracts of Corynebacterium sp. B6. 6. L-Serine dehydratase, purified from Corynebacterium sp. B6 to a specific activity of about 4mumol of product formed/min per mg of protein, was found to exhibit sigmoid kinetics with an [S]0.5 of about 20mM and h identical to 1.4. Similar results were obtained with enzyme preparations from all isolates. The enzyme required Mg2+ for maximum activity, was different from the L-threonine dehydratase also detectable in extracts, and was induced by growth on L-threonine or glycine.     

Highly selective L-threonine 3-dehydrogenase from Cupriavidus necator and its use in determination of L-threonine

l-Threonine level in blood plasma is a biomarker of some diseases and nitrogen imbalance in the body. The determination of l-threonine is interesting and is required for diagnosis and management of inherited metabolic disorder. This is the first report of the specific enzymatic determination of l-threonine by a newly discovered l-threonine 3-dehydrogenase (ThrDH, EC 1.1.1.103) from Cupriavidus necator NBRC 102504. ThrDH, a key enzyme in l-threonine catabolism in microorganisms and animals, catalyzes the NAD(+)-dependent oxidation of l-threonine to 2-amino-3-oxobutyrate. ThrDH from C. necator was purified to homogeneity and fully characterized. l-Threonine and dl-2-amino-3-hydroxyvalerate are the only substrates for ThrDH among other l-amino acids, alcohols, and amino alcohols. The primary amino acid structure of ThrDH belongs to the extended short-chain alcohol dehydrogenase superfamily and is related to GDP-mannose-3',5'-epimerase (GME) from Arabidopsis thaliana. Both enzymes have a glycine-rich NAD(+)-binding domain at the N terminal and conserved catalytic triad of YxxxK residues, but substrate-binding residues of GME were not found in the ThrDH sequence. ThrDH significantly differs from known bacterial and archaea ThrDHs that belong to zinc-binding medium chain alcohol dehydrogenase because of low sequence similarity and the lack of a zinc-binding domain in the sequence. A specific, quantitative, and sensitive enzymatic endpoint method for l-threonine determination was developed by using a ThrDH microplate assay. The assay was successfully applied for determination of l-threonine in human serum and plasma. Our specific determination is simple, convenient, inexpensive, accurate, and suitable for mass screening determination of l-threonine in a number of samples.     

Highly selective l-threonine 3-dehydrogenase from Cupriavidus necator and its use in determination of l-threonine

l-Threonine level in blood plasma is a biomarker of some diseases and nitrogen imbalance in the body. The determination of l-threonine is interesting and is required for diagnosis and management of inherited metabolic disorder. This is the first report of the specific enzymatic determination of l-threonine by a newly discovered l-threonine 3-dehydrogenase (ThrDH, EC 1.1.1.103) from Cupriavidus necator NBRC 102504. ThrDH, a key enzyme in l-threonine catabolism in microorganisms and animals, catalyzes the NAD⁺-dependent oxidation of l-threonine to 2-amino-3-oxobutyrate. ThrDH from C. necator was purified to homogeneity and fully characterized. l-Threonine and dl-2-amino-3-hydroxyvalerate are the only substrates for ThrDH among other l-amino acids, alcohols, and amino alcohols. The primary amino acid structure of ThrDH belongs to the extended short-chain alcohol dehydrogenase superfamily and is related to GDP-mannose-3′,5′-epimerase (GME) from Arabidopsis thaliana. Both enzymes have a glycine-rich NAD⁺-binding domain at the N terminal and conserved catalytic triad of YxxxK residues, but substrate-binding residues of GME were not found in the ThrDH sequence. ThrDH significantly differs from known bacterial and archaea ThrDHs that belong to zinc-binding medium chain alcohol dehydrogenase because of low sequence similarity and the lack of a zinc-binding domain in the sequence. A specific, quantitative, and sensitive enzymatic endpoint method for l-threonine determination was developed by using a ThrDH microplate assay. The assay was successfully applied for determination of l-threonine in human serum and plasma. Our specific determination is simple, convenient, inexpensive, accurate, and suitable for mass screening determination of l-threonine in a number of samples.     

Metabolism of 2-oxoaldehydes in yeasts. Possible role of glycolytic bypath as a detoxification system in L-threonine catabolism by Saccharomyces cerevisiae

L-Threonine catabolism by Saccharomyces cerevisiae was studied to determine the role of glycolytic bypath as a detoxyfication system of 2-oxoaldehyde (methylglyoxal) formed from L-threonine catabolism. During the growth on L-threonine as a sole source of nitrogen, a large amount of aminoacetone was accumulated in the culture. The enzymatic analyses indicated that L-threonine was converted into either acetaldehyde and glycine by threonine aldolase or 2-aminoacetoacetate by NAD-dependent threonine dehydrogenase. Glycine formed was condensed with acetyl-CoA by aminoacetone synthase to form 2-aminoacetoacetate, a labile compound spontaneously decarboxylated into aminoacetone. The enzyme activities of the glycolytic bypath of the cells grown on L-threonine were considerably higher than those of the cells grown on ammonium sulfate as a nitrogen source. The result indicated the possible role of glycolytic bypath as a detoxification system of methylglyoxal formed from L-threonine catabolism.     

Growth, enzyme levels, and some metabolic properties of an Escherichia coli mutant grown on L-threonine as the sole carbon source.

A mutant of Escherichia coli (designated E. coli SBD-76) that utilizes L-threonine as the sole carbon source was isolated. In contrast with levels in extracts of wild-type cells, the levels of threonine dehydrogenase in extracts of this mutant were 100-fold higher than levels of threonine aldolase or degradative threonine dehydratase. Catabolite repression of threonine dehydrogenase was manifested in wild-type, but not SBD-76, cells. For purposes of isolating enzymes, large quantities of SBD-76 cells with the elevated threonine dehydrogenase level could be grown in a fermentor in modified Fraser medium containing 1% glycerol, rather than in the 0.2% L-threonine minimal medium used to isolate the mutant. SBD-76 cells grown on L-threonine excreted glycine and aminoacetone into the medium, and extracts of the mutant strain catalyzed a quantitative conversion of L-threonine to glycine and aminoacetone.     

Metabolism of threonine by Fusaria growth on threonine as the sole carbon and nitrogen source

Three strains ofFusarium supporting aerobic growth onl-threonine as the sole source of energy and carbon and nitrogen, initially metabolised threonine to acetyl-CoA and glycine via induciblel-threonine:NAD⁺ dehydrogenase plus 2-amino-3-oxobutyrate:CoA ligase activities. Comparative enzyme induction patterns after growth of the three strains on a wide range of carbon sources indicated that the glycine produced by the NAD⁺ plus CoASH-dependent cleavage of threonine was subsequently utilised as an energy source and biosynthetic precursor via the glycine-serine pathway, pyruvate carboxylase, and ultimately the TCA cycle. Acetyl-CoA, the second initial C₂ threonine catabolism product, was subsequently assimilated via a combined TCA plus glyoxylate cycle.     

Factors regulating gluconeogenesis in chick embryo liver]

The ratio NAD+/NADH in cytoplasm and mitochondria of chicken embryo liver does not change up to the stage of hatching. After the hatching this ratio decreases 2-fold in both cytoplasm and mitochondria. The hatching is also accompanied by the decrease of total and mitochondrial contents of oxaloacetate and of oxaloacetate/malate ratio, the activity of citrate synthase and the ratio acetyl-CoA/CoA being unchanged.     

Biosynthesis of amino acids in Clostridium pasteurianum

1. Clostridium pasteurianum was grown on a synthetic medium with the following carbon sources: (a) (14)C-labelled glucose, alone or with unlabelled aspartate or glutamate, or (b) unlabelled glucose plus (14)C-labelled aspartate, glutamate, threonine, serine or glycine. The incorporation of (14)C into the amino acids of the cell protein was examined. 2. In both series of experiments carbon from exogenous glutamate was incorporated into proline and arginine; carbon from aspartate was incorporated into glutamate, proline, arginine, lysine, methionine, threonine, isoleucine, glycine and serine. Incorporations from the other exogenous amino acids indicated the metabolic sequence: aspartate --> threonine --> glycine right harpoon over left harpoon serine. 3. The following activities were demonstrated in cell-free extracts of the organism: (a) the formation of aspartate by carboxylation of phosphoenolpyruvate or pyruvate, followed by transamination; (b) the individual reactions of the tricarboxylic acid route to 2-oxoglutarate from oxaloacetate; glutamate dehydrogenase was not detected; (c) the conversion of aspartate into threonine via homoserine; (d) the conversion of threonine into glycine by a constitutive threonine aldolase; (e) serine transaminase, phosphoserine transaminase, glycerate dehydrogenase and phosphoglycerate dehydrogenase. This last activity was abnormally high. 4. The combined evidence indicates that in C. pasteurianum the biosynthetic role of aspartate and glutamate is generally similar to that in aerobic and facultatively aerobic organisms, but that glycine is synthesized from glucose via aspartate and threonine.     

Three-dimensional structure of 2-amino-3-ketobutyrate CoA ligase from Escherichia coli complexed with a PLP-substrate intermediate: inferred reaction mechanism

2-Amino-3-ketobutyrate CoA ligase (KBL, EC 2.3.1.29) is a pyridoxal phosphate (PLP) dependent enzyme, which catalyzes the second reaction step on the main metabolic degradation pathway for threonine. It acts in concert with threonine dehydrogenase and converts 2-amino-3-ketobutyrate, the product of threonine dehydrogenation by the latter enzyme, with the participation of cofactor CoA, to glycine and acetyl-CoA. The enzyme has been well conserved during evolution, with 54% amino acid sequence identity between the Escherichia coli and human enzymes. We present the three-dimensional structure of E. coli KBL determined at 2.0 A resolution. KBL belongs to the alpha family of PLP-dependent enzymes, for which the prototypic member is aspartate aminotransferase. Its closest structural homologue is E. coli 8-amino-7-oxononanoate synthase. Like many other members of the alpha family, the functional form of KBL is a dimer, and one such dimer is found in the asymmetric unit in the crystal. There are two active sites per dimer, located at the dimer interface. Both monomers contribute side chains to each active/substrate binding site. Electron density maps indicated the presence in the crystal of the Schiff base intermediate of 2-amino-3-ketobutyrate and PLP, an external aldimine, which remained bound to KBL throughout the protein purification procedure. The observed interactions between the aldimine and the side chains in the substrate binding site explain the specificity for the substrate and provide the basis for a detailed proposal of the reaction mechanism of KBL. A putative binding site of the CoA cofactor was assigned, and implications for the cooperation with threonine dehydrogenase were considered.     

Enzymes of l-(+)-3-hydroxybutyrate metabolism in the rat

The three enzymes required for the production and utilization of l-(+)-3-hydroxybutyrate were sought in various tissues of the rat. All tissues examined contained substantial amounts of (No. 1) l-(+)-3-hydroxybutyryl CoA dehydrogenase (EC 1.1.1.35). The specific activity of (No. 2) l-(+)-3-hydroxybutyryl CoA deacylase (EC 3.1.2) was highest in liver (3.8 mU/mg in mitochondrial matrix (1 U = 1 μmol/min). Brain, heart, and skeletal muscle contained < 20% of this activity. The chromatography of liver mitochondrial “matrix” preparations on DEAE-cellulose resolved the deacylase into two peaks. Peak I hydrolyzed 2- or 3- carbon acylCoA esters more efficiently than l-(+)-3-hydroxybutyrate CoA, while Peak II activity was highest using l-(+)-3-hydroxybutyryl CoA. The K_m(app) for Peak II deacylase with l-(+)-3-hydroxybutyryl CoA was 19 μm. Acyl CoA synthetase (EC 6.2.1.2) (No. 3) was assayed with sorbate (sorboyl CoA ligase) or l-(+)-3-hydroxybutyrate (l-(+)-3-hydroxybutyryl CoA ligase). The highest specific activity for l-(+)-3-hydroxybutyryl CoA ligase was associated with brain mitochondria (8.3 mU/mg). In the “matrix” fraction of rat liver mitochondria the activities of these two acyl CoA synthetases were distinguished chromatographically and by their stability at various pH values. Heart and skeletal muscle mitochondria contained <10% of the liver activities of both ligases. These data implicate the liver as a site of l-(+)-3-hydroxybutyrate production.