Acetyl-CoA hydrolase activity and function in Ascaris suum muscle mitochondria.

Acyl-CoA compounds are stable in adult Ascaris suum mitochondrial preparations. However, when incubated in the presence of 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB), acetyl-CoA or propionyl-CoA are hydrolyzed to form free coenzyme A. This acetyl-CoA hydrolase activity has been partially purified and found to be specific for the above CoA derivatives. Gel filtration indicates an apparent molecular weight of 232,000. The hydrolase activity has been purified free from acyl-CoA transferase activities and appears not to be accounted for on the basis of a thiolase. Because Ascaris is an intestinal parasite that metabolizes primarily anaerobically and accumulates a large number of volatile fatty acids that are formed as the coenzyme A derivatives, the hydrolase would be expected to function in the regeneration of free CoA. However, how the hydrolase reaction would be pulled in the absence of the nonphysiologic DTNB is not known.

Acyl-CoA transferase activities in homogenates of Fasciola hepatica adults.

Succinyl-CoA is an intermediate in the formation of the fermentation product, propionate, by Fasciola hepatica adults. Acyl-CoA transferase activities are present in crude homogenates of Fasciola, which could account for the synthesis of succinyl-CoA from succinate by the transfer of CoA from either propionyl-CoA or acetyl-CoA. No transferase activity was apparent from 2-methylbutyryl-CoA or 2-methylvaleryl-CoA as was previously reported for the nematode, Ascaris suum. Heat denaturation experiments indicate that all of the Fasciola transferase activities may result from a single protein.

Propionyl-CoA condensing enzyme from Ascaris muscle mitochondria. I. Isolation and characterization of multiple forms.

The condensation of two propionyl-CoA units or a propionyl-CoA with acetyl-CoA is required for the synthesis of 2-methylvalerate or 2-methylbutyrate, respectively, two of the major fermentation products of Ascaris anaerobic muscle metabolism. An enzyme that preferentially catalyzes the condensation of propionyl-CoA rather than acetyl-CoA has been purified from the mitochondria of the parasitic intestinal nematode Ascaris lumbricoides var. suum. The purified enzyme is over 10 times more active with propionyl-CoA than with acetyl-CoA as substrate. It also catalyzes the coenzyme A-dependent hydrolysis of acetoacetyl-CoA at a rate four times higher than the propionyl-CoA condensation reaction. The purified Ascaris condensing enzyme preferentially forms the 2-methyl-branched-chain keto acids rather than the corresponding straight chain compounds. The native molecular weight of the purified enzyme was estimated to be 160,000 by gel filtration chromatography and 158,000 by high pressure liquid chromatography. The enzyme migrated as a single protein band with Mr 40,000 during sodium dodecyl sulfate-polyacrylamide electrophoresis, indicating that the enzyme is composed of four subunits of the same molecular weight. Chromatography on CM-sephadex resulted in the isolation of two separate peaks of activity, designated as A and B. Both A and B had the same molecular weight and subunit composition. However, they differed in their specific activities and isoelectric points. The pIs of condensing enzymes A and B were 7.6 and 8.4, respectively. Propionyl-CoA was the best substrate for the condensation reaction with both enzymes. However, the specific activity of enzyme B for both propionyl-CoA condensation (3.4 mumol/min/mg protein) and acetoacetyl-CoA thiolysis (13.8 mumol/min/mg protein) was 2.4 times higher than that obtained with enzyme A. Similarly, chromatography on phosphocellulose resolved the Ascaris condensing enzyme activity into one minor and two major peaks. All of these components had the same molecular weight and subunit composition, but differed in their specific activities. The two major phosphocellulose peaks cross-reacted immunologically when examined by the Ouchterlony double immunodiffusion technique. In addition, antiserum against the phosphocellulose most active form cross-reacted with forms A and B isolated by chromatography of the enzyme on CM-Sephadex, indicating that all forms were immunochemically related.

Propionyl-CoA condensing enzyme from Ascaris muscle mitochondria. II. Coenzyme A modulation.

The propionyl-CoA condensing enzyme which catalyzes the first step in the biosynthesis of 2-methylbutyrate and 2-methylvalerate by Ascaris muscle appears to exist in at least three forms in the mitochondria of this parasitic nematode. Two forms, A and B, were separated by ion exchange chromatography on CM-Sephadex. Chromatography on phosphocellulose resulted in the recovery of one minor peak (I) and two major peaks with activity (II and III). A and B as well as I, II, and III differed in their specific activities. Forms B and III were the most retained by their resins, and were the most active forms of the enzyme in each case. Inhibition studies with metabolites from Ascaris mitochondria indicate that CoASH, a product of the condensation reaction, and acetyl-CoA are effective inhibitors of the condensing and thiolytic activities of the Ascaris enzyme, respectively. Incubation of the active enzyme form B for 2 h with 0.1 mM CoASH at room temperature under nitrogen caused the loss of 92% of the propionyl-CoA condensing activity and 67% of the thiolase activity when assayed in standard mixtures. The propionyl-CoA condensing enzyme exhibited a hyperbolic dependence of the condensation velocity to changes in substrate concentration. However, in the presence of CoASH the Michaelis-Menten kinetics was transformed into a sigmoidal kinetics indicating a deviation from a simple product inhibition. Inactivation of the most active forms of the enzyme with CoASH was accompanied by (a) a change in the chemical reactivity of the protein toward p-chloromurcuribenzoate, (b) a change in the uv-visible spectrum of the protein, and (c) a change in the elution patterns from both CM-Sephadex and phosphocellulose column chromatography, where-upon one, two, or more protein peaks were obtained. The several protein peaks resolved by rechromatography of the [14C]CoASH-inactivated enzyme III on phosphocellulose had different CoASH contents. The elution positions were correlated with the less active forms (I and II) having increased [14C]CoASH activities. Similarly, the two peaks isolated upon rechromatography of the CoASH-partially inactivated enzyme B on CM-Sephadex had different isotope contents and the elution position of enzyme A corresponded to the less active form. The results described indicate that the CoASH modification of Ascaris propionyl-CoA condensing enzyme may be responsible for the existence of several forms of the enzyme and might represent a mode of control by chemically modulating the amount of the active forms of the enzyme.

2-Methylacetoacetyl-coenzyme A reductase from Ascaris muscle: purification and properties.

2-Methylacetoacetyl-CoA and 3-keto-2-methyl pentanoyl-CoA have been proposed to be intermediates in the synthesis of 2-methylbutyrate and 2-methylvalerate, respectively, by Ascaris lumbricoides muscle. These volatile acids are major fermentation products of Ascaris metabolism. 2-Methylacetoacetyl-CoA reductase has been purified 532-fold from Ascaris muscle to yield a homogeneous preparation which contained a single protein species as observed on discontinuous polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purification procedure utilized subcellular fractionation, affinity chromatography on NAD+ agarose, and ion-exchange chromatography on DEAE-cellulose. A constant activity ratio for ethyl 2-methylacetoacetate and acetoacetyl-CoA was observed during purification, indicating that the same enzyme catalyzed both reactions. In addition, the purified protein catalyzed the NADH-dependent reduction of ethyl-3-keto-2-methyl pentanoate at essentially the same rate as it did ethyl 2-methylacetoacetate. The purified enzyme is a basic protein with an isoelectric point of 8.45 at 4 degrees C. The molecular weight of the native protein (Mr = 64,000 by exclusion chromatography) and the size of the subunit (Mr = 30,000 by dodecyl sulfate-polyacrylamide electrophoresis) indicate that the enzyme is composed of two subunits of the same molecular weight. Substrate-specificity studies, undertaken with the purified protein, demonstrated that the ethyl esters can substitute for the coenzyme A derivatives but this substitution results in an active substrate only when a branched 2-methyl group is present. The straight-chain ethyl ester is inactive. Kinetic constants for the substrates and nucleotides were determined. The role of the CoA esters as the physiological substrates for the Ascaris enzyme is substantiated. When assayed in the reductive direction with ethyl 2-methylacetoacetate as substrate, the activity of the purified enzyme was inhibited not only by coenzyme A as previously reported, but also by acetyl-CoA. The physiological implications of these inhibitions are discussed.

Formyl-methenyl-methylenetetrahydrofolate synthetase(combined) from yeast. Biochemical characterization of the protein from an ade3 mutant lacking the formyltetrahydrofolate synthetase function.

A protein from Saccharomyces cerevisiae mutant ade3-1050, a formyltetrahydrofolate synthetase-deficient mutant, has been purified to apparent homogeneity. The purified mutant enzyme shows both methylenetetrahydrofolate dehydrogenase and methenyltetrahydrofolate cyclohydrolase activities, but lacks formyltetrahydrofolate synthetase activity. The biochemical characterization of the mutant protein described in this paper is consistent with genetic data which indicate that the 1050 mutation is a point mutation at the ade3 locus of chromosome VII of S. cerevisiae. The molecular weight of the native mutant protein (Mr = 227,000 by exclusion chromatography), as well as the number and size of its subunits are exactly the same as those of the trifunctional wild type enzyme. In addition, both proteins have the same sedimentation behavior in a glycerol density gradient (s20,w = 9.4 S), and their activities and structures are equally affected by exposure to mild tryptic degradation. ATP protects both enzymes from tryptic degradation, but NADP+ does not. Some of the kinetic properties of the activities of both enzymes were also determined and were essentially similar. Although both enzymes require the presence of metals for maximal synthetase and dehydrogenase activities, metals are not necessary to maintain their structures intact.