isovaleryl-CoA Research Articles

Purification and properties of short chain acyl-CoA, medium chain acyl-CoA, and isovaleryl-CoA dehydrogenases from human liver.

Short chain acyl-CoA (SCA), medium chain acyl-CoA (MCA), and isovaleryl-CoA (IV) dehydrogenases were purified to homogeneity from human liver using ammonium sulfate fractionation followed by DEAE-Sephadex A-50, hydroxyapatite, Matrex Gel Blue A, agarose-hexane-CoA, and Bio-Gel A-0.5 column chromatographies. The specific activities of the final preparations were enriched 507-, 750-, and 588-fold over those from the second ammonium sulfate fractionation step. The native molecular weights were estimated to be 168,000, 178,000, and 172,000, respectively, by gel filtration. Each of them exhibited, on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, a single protein band with molecular weights of 41,000, 44,000, and 42,000, respectively, indicating a homotetrameric structure. UV/visual spectra, fluorescence spectra, and other evidence indicated that each contains 1 mol of FAD per subunit. They all utilized electron transfer flavoprotein (ETF) or phenazine methosulfate (PMS) as an electron acceptor. The products of SCA dehydrogenase/butyryl-CoA, MCA dehydrogenase/octanoyl-CoA, and IV dehydrogenase/isovaleryl-CoA reactions were identified as crotonyl-CoA, 2-octenoyl-CoA, and 3-methylcrotonyl-CoA, respectively, using gas chromatography. Kinetic parameters Vappmax and Kappm) of these enzymes for various acyl-CoA substrates, as well as Kappm values for ETF and PMS are presented. In general, the substrate specificities of human SCA, MCA, and IV dehydrogenases are slightly less stringent than those of their rat counterparts and resemble those of their bovine and porcine counterparts. The pattern of substrate specificity for these enzymes determined using ETF as electron acceptor significantly differed from that determined using PMS. All of them were severely inhibited by (methylenecyclopropyl)acetyl-CoA.

EFFECTS OF SUBLETHAL DOSES OF ENDOTOXIN AND ASPIRIN (ASA) ON RAT HEPATIC ENERGY METABOLISM: AN ANIMAL MODEL OF REYE SYNDROME

The administration of a sublethal dose of endotoxin (LPS) to fasted rats results in biochemical and histopathological perturbations similar to Reye Syndrome (RS), ie. hyperammonemia, elevated serum lactate and free fatty acids, and hepatic microvesicular fat deposition and swollen mitochondria in the absence of cellular necrosis (Yoder et al, Infect. Immun., 1985). In this study, hepatic energy metabolism was assessed in freeze clamped liver samples (12 hrs post treatment) obtained from (250-300 gm Sprague-Dawley) rats which had received an intraperitoneal injection of placebo-5% dextrose (C), 0.25mg LPS/kg (LPS), placebo + 1hr later 50mg ASA/kg (ASA) or 0.25mg LPS/kg + 1hr later 50 mg ASA/kg (LPS/ASA). In both LPS and LPS/ASA, ATP/ADP declined by 31% (p<0.005), [total ketones] decreased by 40% (p<0.0005) and [acetyl CoA] declined by 37% (p<0.005) as compared to C and ASA. In LPS/ASA, there were increases in [isovaleryl CoA], [isobutyryl CoA] and [B-methylcrotonyl CoA] as compared with ASA (60%, p<0.01) and LPS (90%, p<.005). These results suggest inhibition of fatty acid oxidation and abnormal branch chain amino acid (BCAA) oxidation and are analogous to data obtained in patients with RS showing hyperammonemia, compromised FA oxidation and accumulation of intermediates of BCAA oxidation. Accumulation of BCAA intermediates with LPS/ASA is a possible mechanism for the potentiation of RS by ASA. These findings provide further biochemical evidence that a sublethal dose of LPS administered to fasted rats produces an animal model of RS, (Supported by NS 17752)

Separation of the intermediates of leucine catabolism by high-performance liquid chromatography

A method has been developed for the separation of leucine, 2-ketoisocaproic acid, isovaleryl CoA, 3-methylcrotonyl CoA, 3-hydroxy-3-methylglutaryl CoA, 3-methylglutaconyl CoA, acetyl CoA, and acetoacetic acid by ion-exchange high-performance liquid chromatography. The analysis requires 180 min. Use of this method to assess the catabolism of radiolabeled leucine in normal cultured human skin fibroblasts shows that these cells do not accumulate CoA esters, but convert leucine mainly to 2-ketoisocaproic acid, glutamate, and hydroxyisovalerate. In the fibroblasts of a patient with maple syrup urine disease, only 2-ketoiscaproic acid is produced from leucine.

Glutaric aciduria type II: in vitro studies on substrate oxidation, acyl-CoA dehydrogenases, and electron-transferring flavoprotein in cultured skin fibroblasts.

The previous biochemical evidence had suggested that glutaric aciduria type II (GA II) is due to deficient dehydrogenation of multiple short-chain acyl coenzyme A's (CoA's), bu the precise biochemical mechanism underlying this disease was unknown. We investigated substrate oxidation and in vitro activities of isovaleryl CoA- and butyryl CoA dehydrogenases as well as that of electron-transferring flavoprotein (ETF) in cultured skin fibroblasts from a patient with GA II. GA II cells have a markedly decreased ability to oxidize [1-14C]butyrate, [2-14C]lysine, and [2,14C]leucine (3, 9, and 9% of control values, respectively). Mitochondrial isovaleryl CoA- and butyryl CoA dehydrogenase activities in GA II cells were determined using a tritium release assay with [2,3-3H] acyl-CoA's as substrate. When an artificial electron acceptor, phenazine methosulfate (PMS) was not added in the assay media, these activities were 108 and 113% of controls, respectively. This represents the normal abilities of the dehydrogenases in GA II cells to bind the substrate and to catalyze tritium exchange between the bound substrate and solvent. When PMS was added to the assay mixture, these activities were 88 and 70% of control values, respectively, indicating that these enzymes can both dehydrogenate their substrates normally and then transfer electrons to an acceptor (PMS). ETF activity in mitochondrial sonic supernatants from GA II cells, as assessed by a newly devised method, was 159% of control values. These observations suggest that the acyl CoA dehydrogenases themselves and ETF are not defective in GA II. Therefore, the deficiency of another common gene product necessary for the function of all the affected acyl CoA dehydrogenases must be sought to explain the etiology of GA II.

Demonstration of a specific mitochondrial isovaleryl-CoA dehydrogenase deficiency in fibroblasts from patients with isovaleric acidemia.

To study the enzymatic basis of isovaleric acidemia, we have developed assay methods for isovaleryl-CoA and butyryl-CoA dehydrogenases that measure the amount of tritium released from the respective [2,3-3H]acyl CoAs. Because assay of these enzymes in human fibroblast homogenates was subject to interference by nonspecific reactions, we have isolated mitochondria from cultured skin fibroblasts by protease treatment, homogenization, and differential centrifugation. By using this assay method with these isolated mitochondria, we have demonstrated a specific deficiency of isovaleryl-CoA dehydrogenase [isovaleryl-CoA: (acceptor) oxidoreductase, EC 1.3.99.10] activity in cultured skin fibroblasts from five patients with isovaleric acidemia. In contrast, mitochondrial butyryl-CoA dehydrogenase [butyryl-CoA: (acceptor) oxidoreductase, EC 1.3.99.2] activity in these cells was preserved at normal levels. These results have been reproduced by using the conventional dye reduction assays. These observations give further support to the hypothesis that isovaleryl CoA is dehydrogenated by a specific enzyme and that isovaleric acidemia is due to a deficiency of this enzyme.

Alpha-Ketoisocaproate stimulates branched-chain amino acid transaminase in kidney and muscle.

We have reported that branched-chain amino acid transaminase (BATase) activity of isolated rat kidney is stimulated by perfusion with alpha-ketoisocaproate (KL). This study examines the mechanism of this effect in kidney and documents that stimulation occurs in intact skeletal muscle. Increased activity was not attributable to synthesis of enzyme because it occurred in the presence of cycloheximide. The in vivo degradation rate of BATase estimated from sequential measurements of activity following intravenous cycloheximide was longer than 90 min, whereas during in vitro perfusion stimulation could be detected within 5 min. Incubation of supernatant from kidney homogenate with KL stimulated BATase; incubation with alpha-keto-beta-methylvalerate (KI), alpha-ketoisovalerate (KV), leucine (leu), or isovaleryl CoA did not. Perfusion of rat hindquarter with KL increased muscle BATase activity; perfusion with acetoacetate, KI, KV, or leu did not. Again, cycloheximide studies indicated a direct effect of KL on muscle BATase. These findings suggest that alpha-ketoisocaproate can increase BATase activity and thus may be involved in regulation of its activity.

Inhibition of glycine synthase by branched-chain α-keto acids: A possible mechanism for abnormal glycine metabolism in ketotic hyperglycinemia

The effect of various amino acid metabolites on glycine oxidation by rat liver homogenate was investigated. Three compounds, α-ketoisovaleric acid, α-ketoisocaproic acid, and α-keto-β-methylvaleric acid, were found to inhibit glycine oxidation by 40–60%. In addition, these compounds also inhibited the glycine-CO 2 exchange reaction, a partial reaction of glycine synthase. The reverse reaction, glycine synthesis, was stimulated 4-fold by these α-keto acids. Pyruvate and α-ketoglutarate had no effect on any of these reactions. The parent amino acids, valine, isoleucine, and leucine, also had no effect on the reactions nor did any of their other metabolites with the exception of the branched-chain α-keto acids. The concentration dependence of the inhibition of glycine oxidation and stimulation of glycine synthesis by these branched-chain α-keto acids suggested that the inhibition of glycine oxidation by these compounds was the result of their further oxidation by branched-chain α-keto acid dehydrogenase. However, the products of the branched-chain α-keto acid dehydrogenase, isobutyryl CoA, isovaleryl CoA, or α-methylbutyryl CoA had no effect on glycine oxidation. Thus, it appeared that either the branched-chain α-keto acids altered glycine oxidation by direct binding to glycine synthase or that electrons derived from the oxidation of branched-chain α-keto acids were transferred to the glycine synthase system. It is proposed that glycine synthase and branched-chain α-keto acid dehydrogenase either share a common subunit, possibly lipoamide dehydrogenase, or are so arranged on the mitochondrial membrane that electron transfer between these two enzymes occurs.

Branched chain 2-oxo-acid dehydrogenase complex of rat liver

It is generally assumed that branched chain 2-oxoacids formed from leucine, isoleucine and valine are oxidised by dehydrogenase complex(es) analogous to pyruvate and 2-oxoglutarate dehydrogenase complexes. There is much recent work on the regulation of oxidation of branched chain amino acids and 2-oxo-acids in vivo but little information on substrate kinetics or regulation by metabolite effectors of the branched chain 2-oxo-acid dehydrogenase complex(es). We describe here a method for extraction and partial purification of a complex from rat liver mitochondria, K m values for substrates and inhibition by isovaleryl CoA (competitive with CoA) and NADH (competitive with NAD÷). Evidence is given that a single complex may oxidise all 3 branched chain 2-oxo-acids.

STUDIES ON KETOTIC HYPERGLYCINEMIA-INHIBITORS OF SERINE HYDROXYMETHYL TRANSFERASE

Recently Hillman and Otto showed tiglic acid to inhibit glycine-serine interconversion in fibroblasts from a patient with β-ketothiolase deficiency and normal cells. In order to elucidate the mechanism of these phenomena, the effects of tiglyl CoA and related compounds on partially purified rabbit liver serine hydroxymethyltransferase (SHMT) were investigated. The rate of conversion of serine to glycine was measured as described by Schirch. Using SHMT 27. 7 units/ml, serine 16mM, and dl-L-tetrahydrofolate 0.32mM, the concentration of tiglyl CoA and % inhibition (N=5, Mean ±S. E. M.) of the enzyme activity were 2.5mM 15±1.5, 5.0mM 28±2.4, 7.5mM 40±2.8, 10mM 41±3.3. Isovaleryl CoA, n-valeryl CoA, crotonyl CoA and isobutyryl CoA also showed inhibitory effects, but at a lesser degree than tiglyl CoA. n-Butyryl CoA, propionyl CoA and CoA inhibited only slightly. Tiglate, isovalerate, n-valerate, crotonate, isobutyrate and β-methylcrotonate at a concentration of 30mM were incapable of inhibiting SHMT activity. Tiglyl CoA inhibition showed competitive kinetics with tetrahydrofolate, but not serine. The findings suggest that impaired glycine-serine metabolism in the ketotic hyperglycinemia syndrome may, at least in part, result from inhibition of SHMT by high intracellular concentration of Tiglyl CoA.

Possibility of inborn defect in isovalericacidaemia involving altered enzyme specificity rather than total inactivity.

TANAKA, Isselbacher and colleagues1 have described an inherited metabolic defect, isovalericacidaemia, which, though not fatal in their patients, leads, if untreated, to mental retardation and episodes of convlusion. As the name implies, the condition is characterised by high levels of isovaleric acid in the plasma. During periods of remission, this compound, which arises from the breakdown of dietary leucine, is excreted as a glycine conjugate. From the fact that only isovaleric acid accumulates and not butyric acid (from fat oxidation) or isobutyric or α-methyl butyric acids (from valine and isoleucine breakdown) Tanaka et al.1 have concluded that there must be a specific isovaleryl CoA dehydrogenase that is non-functional in isovalericacidaemia. It had been assumed previously2 that straight and branched-chain fatty acyl CoA compounds alike were oxidised by the green (that is, short-chain) acyl CoA dehydrogenase (see Fig. 1).

Biosynthesis of triacylglycerols containing isovaleric acid

Summary Melon tissues from the porpoise ( Tursiops truncatus ) were incubated with L-(U-14C) leucine in Krebs-Henseleit buffer solution fortified with glucose. Rapid decarboxylation of the radioactively labelled L-leucine was accompanied by extensive incorporation of radioactivity into isovaleroyl chains of triacylglycerols. Relatively little radioactivity was incorporated into long-chain acids. The findings show that isovaleryl CoA, a catabolite of L-leucine, is directly incorporated into lipid structure to a significant degree instead of being degraded to acetate and acetoacetate by the pathway characteristic of other mammalian tissues.

Hypoglycin A: a specific inhibitor of isovaleryl CoA dehydrogenase.

Evidence is presented for the specific in vivo and in vitro inhibition of isovaleryl CoA dehydrogenation by hypoglycin A and its derivative, alpha-ketomethylenecyclopropylpropionic acid. alpha-Methylbutyryl CoA dehydrogenation was also impaired, but the degree of inhibition was much lower. Isobutyryl CoA dehydrogenation was not inhibited. 4-Pentenoic acid inhibited none of these reactions. It is concluded that isovaleryl CoA is dehydrogenated by a specific enzyme, isovaleryl CoA dehydrogenase, contrary to previous assumptions that it is dehydrogenated by green acyl CoA dehydrogenase. The present concept agrees with our previous findings in isovaleric acidemia, a genetic disorder in which a specific defect of isovaleryl CoA dehydrogenase was observed. It was also demonstrated that isovaleric acidemia can be induced in experimental animals by the administration of hypoglycin A. Furthermore, some symptoms of "the vomiting sickness of Jamaica" appear to be due to isovaleric acid accumulation secondary to the ingestion of hypoglycin A.