Effects of amino acids on Thiobacillus acidophilus. I. Growth studies with special reference to valine.

The heterotrophic growth of Thiobacillus acidophilus was inhibited by branched-chain amino acids; valine, isoleucine, and leucine. The inhibition by valine and leucine were partially reversed by isoleucine, and the inhibition by isoleucine was partially reversed by valine. Inhibitions by methionine or threonine were partially reversed when both amino acids were present in the growth medium. Inhibition by tyrosine was increased by phenylalanine or tryptophan. Cystine completely inhibited growth. Other amino acids tested produced little or no inhibition. Acetohydroxy acid synthetase (AHAS) activity was demonstrated in crude extracts of T. acidophilus. In crude extracts the optimum pH was 8.5 with a shift to 9.0 in the presence of valine. Valine was the only branched-chain amino acid which inhibited the AHAS activity. The presence of only one peak of AHAS activity upon centrifugation in linear glycerol density gradients demonstrated that the AHAS activity sediments as one component.     

Biosynthesis of Branched-Chain Amino Acids in Schizosaccharomyces pombe: Properties of Acetohydroxy Acid Synthetase1

The regulatory properties of acetohydroxy acid synthetase (AHAS), the first enzyme in the biosynthetic pathway to valine and the second in the isoleucine pathway, were investigated in the fission yeast Schizosaccharomyces pombe. The enzyme was partially purified from crude extracts by protamine sulfate treatment, ammonium sulfate fractionation, and gel filtration through Sephadex G-25. AHAS from S. pombe is unique in that its activity shows a single peak around pH 6.5; high sensitivity to feedback inhibition by valine at this pH (K(i) = 0.1 mM) indicates that the enzyme is involved in valine biosynthesis. Pyruvate saturation kinetics of AHAS extracted from cells grown on glycerol as sole carbon and energy source were normal and hyperbolic. In contrast, the enzyme from glucose-grown cells exhibited sigmoidal saturation kinetics, an effect which disappeared when the synthetase from such cells was partially purified. This phenomenon was shown to be due to competition for pyruvate between AHAS and pyruvate decarboxylase; the latter enzyme is present in large amounts in cells fermenting glucose. Valine inhibition is noncompetitive in nature, and this effector exhibits homotropic cooperative effects; isoleucine is a less-potent inhibitor of AHAS activity. Mercurial treatment reversibly desensitized the enzyme to valine inhibition. On the basis of these data, the S. pombe AHAS appears to be an allosteric regulatory enzyme with the properties of a negative V system.     

Identification and partial characterisation of a chitinase from Nile tilapia, Oreochromis niloticus

Measurement of chitinase activity in extracts from stomach, intestine, and serum of Nile tilapia with the artificial substrates 4-methylumbelliferil beta-D-N,N'-diacetylchitobioside and 4-methylumbelliferil beta-D-N,N'N"-triacetylchitotrioside (4MU[GlcNAc](2,3)) showed that an endochitinase was involved in the liberation of the fluorophore 4-methylumbelliferone (MU). Enzymes were isolated from tilapia serum by a combination of gel filtration, ion exchange, and reverse-phase chromatography. The molecular mass of the enzyme was estimated to be 75 kDa by SDS-PAGE, suggesting that the enzyme occurs as a monomer. The partially purified enzyme showed maximal activity at pH 7.0 when assayed with 4MU[GlcNAc](2) and lost its activity below pH 5.0 and above pH 8.0. The optimal pH of the purified enzyme toward the substrate 4MU[GlcNAc](3) was pH 9.0 and activity was lost below pH 8.0 and above pH 9.0. Our study has revealed the presence of a chitinolytic enzyme in the gastrointestinal tract and serum that may play a role in digestion and/or defense.     

An improved assay for hepatic glycogen synthase in liver extracts with emphasis on synthase R

An assay for measurement of optimal amounts of glycogen synthase R, the physiologically active form of the enzyme, in liver tissue extracts is described. Tissue extracts enriched in synthase R had a pH profile different from those reported for synthase D and synthase I. In tissue extracts, synthase I had a broad pH optimum but maximal activity was present at pH 7.0-9.0. Synthase D had a sharp pH optimum at pH 8.5 and had little activity at pH 7.0, either in the presence or in the absence of glucose 6-phosphate (G6P). In extracts enriched in synthase R, the pH optimum was 7.0-8.0 without G6P, but 8.0 with G6P. The synthase R activity without G6P rapidly decreased at a higher pH. The proportion of synthase in the physiologically active form traditionally has been reported as an activity ratio based upon the activity in the presence and absence of G6P. The assay has been performed at a single pH. Because of the differences in pH profile, we recommend that the enzyme be measured at pH 7.0 in the absence of G6P and pH 8.5-8.8 in the presence of G6P. In previous assays the substrate UDP-Glc concentration used often has been less than saturating, and the G6P concentration generally has been excessive. A substrate concentration of 11 mM UDP-Glc was found to be necessary for maximal activity. A G6P concentration of 2 mM is adequate for measurement of the D form of the enzyme.     

Regulation of phosphoenolpyruvate carboxylase from maize leaves by nitrate and alanine.

Nitrate and alanine were found to stimulate partially purified maize leaf phosphoenolpyruvate carboxylase under specific assay conditions. Both metabolites stimulated the enzyme at low pH (7.0-7.5) and low substrate levels (1mM phosphoenolpyruvate). Nitrate was found to have a biphasic effect on the enzyme, stimulating at low concentrations (1mM-3mM), with a decrease in stimulation at higher levels. Nitrate caused inhibition of activity at pH 8.0 and although alanine caused some stimulation in activity at pH 8.0 this was not as marked as at the lower pH levels.     

Purification and characterization of polyethylene glycol dehydrogenase involved in the bacterial metabolism of polyethylene glycol

Polyethylene glycol (PEG) dehydrogenase in crude extracts of a PEG 20,000-utilizing mixed culture was purified 24 times by precipitation with ammonium sulfate, solubilization with laurylbetaine, and chromatography with diethylamino-ethyl-cellulose, hydroxylapatite, and Sephadex G-200. The purified enzyme was confirmed to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme, which appeared to consist of four identical subunits, was 2.4 X 10(5). The enzyme was stable below 35 degrees C and in the pH range of 7.5 to 9.0. The optimum pH and temperature of the activity were around 8.0 and 60 degrees C, respectively. The enzyme did not require any metal ions for activity and oxidized various kinds of PEGs, among which PEG 6,000 was the most active substrate. The apparent Km values for tetraethylene glycol and PEG 6,000 were about 10.0 and 3.0 mM, respectively.     

Purification and characterization of polyethylene glycol dehydrogenase involved in the bacterial metabolism of polyethylene glycol.

Polyethylene glycol (PEG) dehydrogenase in crude extracts of a PEG 20,000-utilizing mixed culture was purified 24 times by precipitation with ammonium sulfate, solubilization with laurylbetaine, and chromatography with diethylamino-ethyl-cellulose, hydroxylapatite, and Sephadex G-200. The purified enzyme was confirmed to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme, which appeared to consist of four identical subunits, was 2.4 X 10(5). The enzyme was stable below 35 degrees C and in the pH range of 7.5 to 9.0. The optimum pH and temperature of the activity were around 8.0 and 60 degrees C, respectively. The enzyme did not require any metal ions for activity and oxidized various kinds of PEGs, among which PEG 6,000 was the most active substrate. The apparent Km values for tetraethylene glycol and PEG 6,000 were about 10.0 and 3.0 mM, respectively.     

Properties and reaction mechanism of C4 leaf pyruvate,Pi dikinase

The properties and reaction mechanism of maize leaf pyruvate,Pi dikinase are described. Km values were determined for the forward reaction substrates, pyruvate, ATP, and Pi, at pH 7.4 and 8.0 and for reverse reaction substrates at pH 7.4. Enzyme activity was almost totally dependent on added monovalent cations in both directions. NH+4 was most effective, with Ka values of about 0.38 mM for the forward reaction and 2 mM for the reverse reaction. K+ also completely activated the enzyme in the forward direction (Ka = 8 mM) but only partially activated in the reverse direction. Na+ had little effect on either reaction. The pH optimum for the forward reaction was about 8.2; the reverse reaction optimum was about 6.9. Maximum activity for the reverse direction was about twice the maximum forward direction rate. From data on the requirements for the ATP-AMP exchange reaction, on the mechanism of inhibition of the forward reaction by PEP, AMP, and PPi, and from the kinetics of the interaction of varying certain substrate pairs, it was concluded that the maize leaf pyruvate,Pi dikinase reaction proceeded by the two-step Bi Bi Uni Uni mechanism. This differs from the mechanism of catalysis by the bacterial enzyme.     

Anion transport in red blood cells and arginine-specific reagents. Interaction between the substrate-binding site and the binding site of arginine-specific reagents

Phenylglyoxal is found to be a potent inhibitor of sulfate equilibrium exchange across the red blood cell membrane at both pH 7.4 and 8.0. The inactivation exhibits pseudo-first-order kinetics with a reaction order close to one at both pH 7.4 and 8. The rate constant of inactivation at 37 degrees C was found to be 0.12 min-1 at pH 7.4 and 0.19 min-1 at pH 8.0. Saturation kinetics are observed if the pseudo-first order rate constant of inhibition is measured as a function of phenylglyoxal concentration. Sulfate ions as well as chloride ions markedly decrease the rate of inactivation by phenylglyoxal at pH 7.4, suggesting that the modification occurs at or near to the binding site for chloride and sulfate. The decrease of the rate of inactivation produced at pH 8.0 by chloride ions is much higher than that produced by sulfate ions. Kinetic analysis of the protection experiments showed that the loaded transport site is unable to react with phenylglyoxal. From the data it is concluded that the modified amino acid(s) residues, presumably arginine, is (are) important for the binding of the substrate anion.     

Identification and Characterization of a Biodegradative Form of Threonine Dehydratase in Senescing Tomato (Lycopersicon esculentum) Leaf

Threonine dehydratase (TD; EC.4.2.1.16) is a key enzyme involved in the biosynthesis of isoleucine. Inhibition of TD by isoleucine regulates the flow of carbon to isoleucine. We have identified two different forms of TD in tomato (Lycopersicon esculentum) leaves. One form, present predominantly in younger leaves, is inhibited by isoleucine. The other form of TD, present primarily in older leaves, is insensitive to inhibition by isoleucine. Expression of the latter enzyme increases as the leaf ages and the highest enzyme activity is present in the old, chlorotic leaves. The specific activity of the enzyme present in older leaves is much higher than the one present in younger leaves. Both forms can use threonine and serine as substrates. Whereas TD from the older leaves had the same Km (0.25 mM) for both substrates, the enzyme from the young leaves preferred threonine (Km = 0.25 mM) over serine (Km = 1.7 mM). The molecular masses of TD from the young and the old leaves were 370,000 and 200,000 D, respectively. High levels of the isoleucine-insensitive form of threonine dehydratase in the older leaves suggests an important role of threonine dehydratase in nitrogen remobilization in senescing leaves.     

Purification and properties of threonine deaminase from the X-1 isolate of the genus Thermus

Threonine deaminase (l-threonine dehydratase EC 4.2.1.16) has been partially purified from a new extreme thermophilic bacterium, Thermus X-1, which is similar to T. aquaticus YT-1. The threonine deaminase of strain X-1 has a maximal rate of reaction at 85 to 90 C and is more thermostable than the threonine deaminase from mesophilic bacteria. The enzyme has an apparent molecular weight of 100,000 to 115,000, a K(m) for l-threonine of 14 mM, a pH optimum of 8.0, and like other threonine deaminases also catalyzes the deamination of serine. However the Thermus X-1 threonine deaminase does not show a strong feedback inhibition by isoleucine. It is suggested that the regulation of the biosynthesis of isoleucine in this extreme theromophile may resemble that reported in Rodospirillum rubrum.     

Isoleucine Auxotrophy Due to Feedback Hypersensitivity of Biosynthetic Threonine Deaminase

Isoleucine-deficient mutants of Salmonella typhimurium were isolated. Three groups of mutants can be discerned by their nutritional requirements and enzyme patterns. (i) Mutants which grow with isoleucine alone are devoid of biosynthetic threonine deaminase (TD). (ii) Mutants growing with isoleucine and valine are devoid of transaminase B. (iii) Mutants growing with either isoleucine or threonine have normal levels of TD. However, the sensitivity of this enzyme to feedback inhibition by isoleucine is greatly enhanced. The inhibitory effect of isoleucine can be counterbalanced by high concentrations of threonine. These results indicate that the production of isoleucine in the mutants is restricted to a low level not sufficient to support the growth of the cells. This hypothesis is confirmed by studies with revertants of an isoleucine-threonine mutant. In nine revertants, wild-type properties of TD have been restored. In four revertants, the hypersensitivity of TD is unchanged, but the strains produce a greatly enhanced quantity of threonine, which is excreted into the culture medium. It follows, that hypersensitivity of TD to inhibition by isoleucine is the cause of the nutritional requirement of isoleucine-threonine mutants.     

Enzymes of the Isoleucine-Valine Pathway in Acinetobacter

Regulation of four of the enzymes required for isoleucine and valine biosynthesis in Acinetobacter was studied. A three- to fourfold derepression of acetohydroxyacid synthetase was routinely observed in two different wild-type strains when grown in minimal medium relative to cells grown in minimal medium supplemented with leucine, valine, and isoleucine. A similar degree of synthetase derepression was observed in appropriately grown isoleucine or leucine auxotrophs. No significant derepression of threonine deaminase or transaminase B occurred in either wild-type or mutant cells grown under a variety of conditions. Three amino acid analogues were tested with wild-type cells; except for a two- to threefold derepression of dihydroxyacid dehydrase when high concentrations of aminobutyric acid were added to the medium, essentially the same results were obtained. Experiments showed that threonine deaminase is subject to feedback inhibition by isoleucine and that valine reverses this inhibition. Cooperative effects in threonine deaminase were demonstrated with crude extracts. The data indicate that the synthesis of isoleucine and valine in Acinetobacter is regulated by repression control of acetohydroxyacid synthetase and feedback inhibition of threonine deaminase and acetohydroxyacid synthetase.     

Cloning, expression, purification, and characterization of biosynthetic threonine deaminase from Escherichia coli

Feedback inhibition of the regulatory enzyme threonine deaminase by isoleucine provides an important level of enzymic control over branched chain amino acid biosynthesis in Escherichia coli. Cloning ilvA, the structural gene for threonine deaminase, under control of the trc promoter results in expression of active enzyme upon induction by isopropyl 1-thio-beta-D-galactoside to levels of approximately 20% of the soluble protein in cell extracts. High level expression of threonine deaminase has facilitated the development of a rapid and efficient protocol for the purification of gram quantities of enzyme with a specific activity 3-fold greater than previous preparations. The catalytic activity of threonine deaminase is absolutely dependent on the presence of pyridoxal phosphate, and the tetrameric molecule is isolated containing 1 mol of cofactor/56,000-Da chain. Wild-type threonine deaminase demonstrates a sigmoidal dependence of initial velocity on threonine concentration in the absence of isoleucine, consistent with a substrate-promoted conversion of the enzyme from a low activity to a high activity conformation. The enzymic dehydration of threonine to alpha-ketobutyrate measured by steady-state kinetics, performed at 20 degrees C in 0.05 M potassium phosphate, pH 7.5, is described by a Hill coefficient, nH, of 2.3 and a K0.5 of 8.0 mM. The negative allosteric effector L-isoleucine strongly inhibits the enzyme, yielding a value for nH of 3.9 and K0.5 of 74 mM whereas enzyme activity is greatly increased by L-valine, which yields nearly hyperbolic kinetics characterized by a value for nH of 1.0 and a K0.5 of 5.7 mM. Thus, these effectors promote dramatic and opposing effects on the transition from the low activity to the high activity conformation of the tetrameric enzyme.     

Characterization of fructose 1,6-bisphosphatase from bakers' yeast

Active nonphosphorylated fructose bisphosphatase (EC 3.1.3.11) was purified from bakers' yeast. After chromatography on phosphocellulose, the enzyme appeared as a homogeneous protein as deduced from polyacrylamide gel electrophoresis, gel filtration, and isoelectric focusing. A Stokes radius of 44.5 A and molecular weight of 116,000 was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate resulted in three protein bands of Mr = 57,000, 40,000, and 31,000. Only one band of Mr = 57,000 was observed, when the single band of the enzyme obtained after polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate was eluted and then resubmitted to electrophoresis in the presence of sodium dodecyl sulfate. Amino acid analysis indicated 1030 residues/mol of enzyme including 12 cysteine moieties. The isoelectric point of the enzyme was estimated by gel electrofocusing to be around pH 5.5. The catalytic activity showed a maximum at pH 8.0; the specific activity at the standard pH of 7.0 was 46 units/mg of protein. Fructose 1,6-bisphosphatase b, the less active phosphorylated form of the enzyme, was purified from glucose inactivated yeast. This enzyme exhibited maximal activity at pH greater than or equal to 9.5; the specific activity measured at pH 7.0 was 25 units/mg of protein. The activity ratio, with 10 mM Mg2+ relative to 2 mM Mn2+, was 4.3 and 1.8 for fructose 1,6-bisphosphatase a and fructose 1,6-bisphosphatase b, respectively. Activity of fructose 1,6-bisphosphatase a was 50% inhibited by 0.2 microM fructose 2,6-bisphosphate or 50 microM AMP. Inhibition by fructose 2,6-bisphosphate as well as by AMP decreased with a more alkaline pH in a range between pH 6.5 and 9.0. The inhibition exerted by combinations of the two metabolites at pH 7.0 was synergistic.     

Threonine Synthetase-Catalyzed Conversion of Phosphohomoserine to α-Ketobutyrate in Bacillus subtilis

An enzyme activity of Bacillus subtilis has been found that catalyzes the dephosphorylation and deamination of phosphohomoserine to alpha-ketobutyrate, resulting in a bypass of threonine in isoleucine biosynthesis. In crude extracts of a strain deficient in the biosynthetic isoleucine-inhibitable threonine dehydratase, phosphohomoserine was converted to alpha-ketobutyrate. Phosphohomoserine conversion to alpha-ketobutyrate was shown not to involve a threonine intermediate. Single mutational events affecting threonine synthetase also affected the phosphohomoserine-deaminating activity, suggesting that the deamination of phosphohomoserine was catalyzed by the threonine synthetase enzyme. It was demonstrated in vivo, in a strain deficient in the biosynthetic threonine dehydratase, that isoleucine was synthesized from homoserine without intermediate formation of threonine.     

Characteristics and identity of obligately aerobic spoilage yeasts from fish silage

Yarrowia (Candida) lipolytica was the predominant organism isolated from the surface film of growth derived from ground hake gurry to which only phosphoric acid was added to give a pH of 4.0. The optimum pH for the crude extracellular protease activity of two distinguishable strains of Y. lipolytica, designated CL1 and CL2, with casein as substrate was 7.0. The optimum temperature of the crude extracellular protease activity from both strains was 50 degrees C. The addition of 2.0% glucose to broth cultures resulted in a significant increase in final cell mass and extracellular protease activity but resulted in a reduction in the units of protease activity per mg of dry cell mass at initial pH values of 5.6 and 7.0 but not an initial pH of 8.0.     

Identification of a pH 6.5 acetohydroxyacid synthetase inBacillus subtilis

Acetohydroxyacid synthetase activity of crude extracts ofBacillus subtilis grown in pH 7.0 minimal medium has a pH optimum of 7.5. However, the activity of extracts of cells grown in minimal medium of pH 6.0 shows a pH optimum of 6.5. Acetate or propionate induces formation of the pH 6.5 activity. Hydroxyapatite chromatography of a crude extract of cells grown in pH 7.0 medium shows one major and one minor peak of enzymatic activity. Both peaks have a pH optimum of 7.5–8.0. However, chromatography of an extract of cells grown in the presence of acetate reveals three peaks of activity: one major peak with a pH optimum of 6.5 and two minor peaks both having a pH optimum of 7.5–8.0.     

Properties of a ribonuclease from Aedes aegypti larvae

1. The properties of a soluble ribonuclease from Aedes aegypti larvae have been compared with ribonuclease activity in adult female tissue. 2. In larval extracts ribonuclease activity was maximal at 40-45 degrees C whereas activity in tissue from adult females was highest at 50 degrees C. 3. Ribonuclease activity that was recovered in a 20-60% ammonium sulfate precipitate was further purified by batch elution from DEAE-Sephacel and from carboxymethylcellulose. 4. Ribonuclease activity in the partially purified fraction was sensitive to EDTA, stimulated by magnesium, had a pH optimum at 9.0 and a Mr of 45,000. 5. Agarose gels containing yeast RNA substrate were used to monitor partial purification of the larval ribonuclease.     

Characteristics and identity of obligately aerobic spoilage yeasts from fish silage

R.E. LEVIN AND R. WITKOWSKI. 1991. Yarrowia (Canadida) lipolytica was the predominant organism isolated from the surface film of growth derived from ground hake gurry to which only phosphoric acid was added to give a pH of 4.0. The optimum pH for the crude extracellular protease activity of two distinguishable strains of Y. lipolytica , designated CL1 and CL2, with casin as substrate was 7.0. The optimum temperature of the crude extracellular protease activity from both strains was 50.C. The addition of 2.0% glucose to broth cultures resulted in a significant increase in final cell mass and extracellular protease activity but resulted in a reduction in the units of protease activity per mg of dry cell mass at initial pH values of 5.6 and 7.0 but not an initial pH of 8.0