Dexter S. Goldman

Enzyme systems in the micobacteria VII. Purification, properties and mechanism of action of the alanine dehydrogenase

Abstract An alanine dehydrogenase (AID) has been purified from cell-free extracts of the H 37 Ra strain of Mycobacterium tuberculosis var. hominis . This enzyme catalyzes the reaction: l -alanine +DPN + ⇌ pyruvate + NH 4 + + PNH . Its sensitivity to certain inhibitors suggests that free sulfhydryl groups are necessary for enzymic activity. In the reductive amination reaction pyruvate and NH 4 + are shown to each affect the K s of the other. A kinetic analysis of the reductive amination reaction shows that the mechanism of action of AID can be described as a modified Theorell-chance mechanism with substrate inhibition. Attempts to show the intermediation of spontaneously-formed imino-propionate were unsuccessful. It is proposed that reductive amination reactions proceed in two steps. The first step is the enzymic formation of enzyme-bound amino acid. This is followed by the enzymic reduction of the imino acid by DPNH to the amino acid.

Enzyme systems in the mycobacteria XIII. Glycine dehydrogenase and the glyoxylic acid cycle

Abstract Cell-free extracts of the H37Ra strain of Mycobacterium tuberculosis contain isocitritase and malate synthetase. These enzymes have been partially purified and shown to be similar to the enzymes previously purified from other bacteria. The synthesis of malate from glyoxylate and CoASAc has been shown to be reversible. A new enzyme, glycine dehydrogenase, is described. This enzyme catalyzes the reductive amination of glyoxylate to glycine; this reaction may represent a significant alternate pathway for the formation of glycine in the absence of transamination.

Direct incorporation of octanoate into long-chain fatty acids by soluble enzymes of Mycobacterium tuberculosis

Abstract The incorporation of acetate into long-chain fatty acids by cell-free extracts of the H37Ra strain of Mycobacterium tuberculosis is markedly stimulated by the presence of other fatty acids; the stimulation is maximal when octanoate is added to the system. A partially-purified enzyme system catalyzes the avidin-insensitive elongation of octanoyl-CoA by acetyl-CoA. In the absence of acetyl-CoA, acyl-CoAs such as octanoyl-CoA or decanoyl-CoA are incorporated directly into fatty acids by what appears to be intermolecular condensations. Thus, [1-14C] octanoyl-CoA yields C16, and and C24 fatty acids with one-half and one-third, respectively, of the 14C in the carboxyl carbon. We suggest the possibility that, in M. tuberculosis, synthesis of long-chain fatty acids may be accomplished through direct condensation of shorter chain length acyl-CoAs rather than only through a stepwise elongation mechanism.

Enzyme systems in the mycobacteria. IX. The reductive acetylation of lipoic acid.

Abstract The pyruvic dehydrogenase complex of the H37Ra strain of Mycobacterium tuberculosis catalyzes the reductive acetylation of lipoic acid. The reaction is inhibited by N-ethyl-maleimide and not by arsenite. CoA does not participate in this reaction. A scheme is presented which attempts to unify these and other findings on the mechanism of the oxidative decarboxylation of pyruvate. If the oxidative decarboxylation of [2- 14 C]pyruvate is carried out in the absence of external oxidizing agents but in the presence of free acetaldehyde, the external acetaldehyde becomes labeled. This indicates that enzyme-bound acetaldehyde (or a compound which rearranges to acetaldehyde when it dissociates from the enzyme) is an intermediate in the oxidative decarboxylation of pyruvate.

Enzyme systems in the mycobacteria. VI. Further studies on the pyruvic dehydrogenase system.

1. 1. The pyruvic dehydrogenase complex of the H37Ra strain of Mycobacterium tuberculosis var. hominis has been resolved into two fractions. 2. 2. The first fraction, precipitating at high ammonium sulfate concentrations, contains a lipoic dehydrogenase. This enzyme catalyzes the oxidation of lip(SH)2 by DPN; the reverse reaction has not been demonstrated. The lipoic dehydrogenase has been purified at least twenty-fold over the crude cell-free extract. DL-α-lipoamide is reduced by DPNH and the lipoicc dehydrogenase. E0′ for the lip(SH)2 ⤥ lipS2 couple at 22° and pH 6.0 was shown to be —0.23 V. 3. 3. The second fraction, precipitating at low ammonium sulfate concentrations, contains, in addition to the pyvuric dehydrogenase, a lipoic transacetylase. This enzyme catalyzes the reversible transfer of an acetyl group from acetyl CoA to lip(SH)2. The enzyme has been purified at least fifteen-fold over the crude cell-free extract. 4. 4. Associated with the pyruvic dehydrogenase-lipoic transacetylase complex is a lipoic deacylase which hydrolytically cleaves S-acetyl-lip(SH) to acetate and lip(SH)2. 5. 5. The possible existence of a soluble pyruvic dehydrogenase complex of enzymes in cell-free extracts of H37Ra and the presence of another form of lipoic acid are discussed.

Enzyme systems in the mycobacteria V. The pyruvic dehydrogenase system

Abstract A soluble pyruvic dehydrogenase has been isolated from cell-free extracts of the H 37 Ra strain of Mycobacterium tuberculosis var. hominis . Pyruvate is oxidatively decarboxylated to acetyl CoA and CO 2 . Magnesium, DPN, CoA and DPT are required for this oxidation. The pyruvic dehydrogenase is inhibited by versene; magnesium can, in part, reverse this inhibition. K S values for DPN and CoA are 1.3·10 −5 and 5.5·10 −5 moles/liter, respectively. The pyruvic dehydrogenase appears to be part of an enzyme complex. Associated with the pyruvic dehydrogenase are a phosphotransacetylase, a lactic dehydrogenase and a lipoic dehydrogenase.

OXIDATION OF REDUCED NICOTINAMIDE-ADENINE DINUCLEOTIDE BY SUBCELLULAR PARTICLES FROM MYCOBACTERIUM TUBERCULOSIS.

Abstract 1. 1. Cell-free extracts of the H 37 Ra strain of Mycobacterium tuberculosis contain a particulate NADH 2 oxidase. The particles have been separated according to size by differential ultracentrifugation. The NADH 2 oxidase specific activity and the cytochrome composition of these particles are the same in all these particles which range from 200 A in diameter to 2000 A in diameter. We conclude that the range in particle size is due to the preparative method employed rather than to any minimal repetitive particle size. 2. 2. One cytochrome each of the a , b and c groups is present, along with flavin, iron and copper, in these particles. The NADH 2 oxidase is unaffected by cyanide. The cytochrome c is quite similar to mammalian cytochrome c 1 . 3. 3. The non-ionic detergent Triton X-100 activates the NADH 2 oxidase activity of these particles. Much of the flavin and protein associated with the particle is removed by this treatment but the cytochrome ratio remains unchanged. We interpret this in terms of a partial “opening” phenomenon. 4. 4. The NADH 2 oxidase particles do not catalyze the oxidation of NADPH 2 , succinate, α-ketoglutarate, pyruvate or β-hydroxybutyrate. Oxygen, indophenol or tetrazolium salts can serve as electron acceptor during the oxidation of NADH 2 .

Intracellular localization of a 6-O-methyl-d-glucose containing soluble polysaccharide from Mycobacterium tuberculosis

Abstract A soluble non-dialyzable low molecular weight (4400) polysaccharide has been purified from the cell-free extract of the H37Ra strain of Myobacterium tuberculosis. The polysaccharide is not covalently bound to other cellular constituents; no hydrolytic procedures are used in its isolation. The polysaccharide is composed of d -glucose (60%) and 6-O- methyl- d -glucose (40%) and one acid group whose location is not yet known. Periodate degradation suggests that most of the 6-O- methyl- d -glucoses serve as branch points in the backbone of the polysaccharide. The polysaccharide is limited in its distribution to the soluble portion of the cytoplasm; 6-O- methyl- d -glucose is absent from hydrolysates of purified preparations of the mycobacterial cell wall, cytoplasmic membranes and ribosomes.

The requirement for a naphtoquinone in the reduced nicotinamide-adenine dinucleotide oxidase system of Mycobacterium tuberculosis☆

Abstract Solvent extraction of sub-cellular particles of the H 37 Ra strain of Mycobacterium tuberculosis inactivates the NADH 2 oxidase system. NADH 2 oxidation can be partially restored by the addition of a lipid factor to the solvent-extracted particles. This factor has been tentatively identified (infrared spectrophotometry and paper chromatography) as vitamin K 2(45) . The activation of the solvent-extracted particles is inhibited by dicumarol; no other compound so far tested significantly inhibits NADH 2 oxidation. The activation of the acetone-extracted particles requires a detergent for maximum effect. Other naphthoquinones and some benzoquinones also activate the system.

Enzyme systems in the mycobacteria: IV. The pyruvic oxidase

Abstract A soluble pyruvic oxidase has been isolated from cell-free extracts of the H37Ra strain of Mycobacterium tuberculosis var. hominis . The enzyme catalyzes the oxidation of pyruvate to acetate; DPT is required for this reaction. Magnesium stimulates the reaction slightly, versene is a strong inhibitor. Ferricyanide, PIP and oxygen can all act as electron acceptors in the activity ratio of 2.5:1:3. No evidence exists that this enzyme can be separated into more than one enzymically active component.