Géza Dénes

Mechanism of arginine biosynthesis in Chlamydomonas reinhardti: II. Purification and properties of N-acetylglutamate 5-phosphotransferase, the allosteric enzyme of the pathway

Abstract 1. 1. The allosteric enzyme of the arginine pathway, N-acetylglutamate 5-phosphotransferase (ATP: N- acetyl - l - glutamate 5-phosphotransferase), which catalyzes the formation of N-acetylglutamate 5-phosphate from N-acetylglutamate and ATP, has been purified 120-fold from cells of the fresh water alga Chlamydomonas reinhardti. 2. 2. The pH optimum of the enzymic reaction is 5.5. The enzyme requires the bivalent ions Mg2+ or Co2+ for activity. The initial velocity data obtained obey the normal Michaelis-Menten kinetics, and do not change in form with variation of pH between 5.5 and 7.5. The apparent Km value of the enzyme at pH 5.5 is 1.5 · 10−2 M for N- acetyl- l -glutamate and 1.6 · 10−3 M for ATP. 3. 3. l -Arginine inhibits the activity of the enzyme, and pH optimum for inhibition is 7.5. The initial velocity data in the presence of l -arginine are compatible with normal Michaelis-Menten kinetics. The kinetic relationship between N- acetyl- l -glutamate and l -arginine is competitive; between ATP and l -arginine it is non-competitive and does not change in form with variation of pH between pH 5.5 and 7.5. The inhibition of enzyme activity by l -arginine is apparently a bimolecular reaction at any pH between pH 5 5 and 7 5. 4. 4. Structural analogs of arginine such as l -canavanine and l -citrulline, in higher concentration as compared with arginine, also inhibit the enzyme activity. 5. 5. Urea at low concentrations reversibly suppresses the inhibitory effect of l -arginine and has no effect on enzyme activity. The kinetic relationship between urea and l -arginine is competitive. 6. 6. l -Arginine protects the enzyme against the inactivating effects of heat and high concentration of urea.

Mechanism of arginine biosynthesis in Chlamydomonas reinhardti I. Purification and properties of ornithine acetyltransferase

Abstract 1. 1. Ornithine acetyltransferase (proposed name, α-N- acetyl- l -ornithine: l -glutamate N-acetyltransferase) which catalyzes the first step in arginine biosynthesis, the formation of N-acetylglutamate from α-N- acetyl- l -ornithine and l-glutamate , has been isolated from the freshwater alga Chlamydomonas reinhardti. 2. 2. The enzyme has a broad pH optimum between 7.5 and 9. The Km value of the enzyme at pH 7.5 is 1.3 · 10−2 M for glutamate and 5.5 · 10 −3 M for α-N- acetyl- l -ornithine . The reaction is reversible; the equilibrium constant expressed as K = [acetylglutamate][ornithine]/[acetylornithine][glutamate] is 0.47. 3. 3. Beside the transferase activity, the enzyme has also a hydrolytic activity. The rate of the hydrolytic reaction for α-N acetylornithine is 1% of that of the acetyltransferase reaction. 4. 4. No specific cofactor has been found. The enzyme is inhibited by p-chloromercuribenzoate, but not by iodoacetate.

Purification and properties of the 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (phenylalanine sensitive) of Escherichia coli K12: II. Inhibition of activity of the enzyme with phenylalanine and functional group-specific reagents

Abstract 1. 1. The inhibition of activity of the phenylalanine-sensitive 3-deoxy- d -arabino-heptulosonate-7-phosphate (DAHP) synthase (7-phospho-2-oxo-3-deoxy- d -arabino-heptonate d -erythrose-4-phosphate-lyase (pyruvate-phosphorylating), EC 4.1.2.15) has been studied. The allosteric effector, l -phenylalanine, inhibits the activity of enzyme noncompetitively for both phosphoenolpyruvate (PEP) and erythrose 4-phosphate. Phenylalanine protects the enzyme against the inactivating effect of heat, and the Ki of enzyme for phenylalanine is 3.8 · 10 5 M. 2. 2. Bromopyruvate, an alkylating structural analog of PEP, irreversibly inactivates the enzyme, and PEP protects the enzyme against the inactivating effect of the alkylating agent. The initial step of the bromopyruvate enzyme interaction obeys the regular saturation kinetics, and the relation between bromopyruvate and PEP is competitive. The Ks of the enzyme for bromopyruvate is 2.0 mM, and the inactivation half-time of the enzyme is 1.5 min. 3. 3. DAHP synthase is a thiol enzyme. p-Hydroxymercuribenzoate reversibly, and dithiobis nitrobenzoic acid irreversibly, inactivates the enzyme. PEP protects the enzyme against the inactivating effect of 5,5′-dithio-bis-(nitrobenzoic acid). 4. 4. Metal-binding agents such as cyanide, EDTA, o-phenanthroline and 1-nitroso-2-naphthol inhibit the activity of the enzyme. The enzyme contains firmly bound heavy metal, probably Co2+.

Purification and properties of the 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (phenylalanine sensitive) of Escherichia coli K12: I. Purification of enzyme and some of its catalytic properties

Abstract 1. 1. The phenylalanine-sensitive allosteric first enzyme (isoenzyme 1a) of the aromatic amino acid biosynthetic pathway, 3- deoxy- d -arabino-heptulosonate-7-phosphate synthase ( 7- phospho-2-oxo-3-deoxy- d -arabino-heptonate d -erythrose-4-phosphate-lyase (pyruvate-phosphorylating), EC 4.1.2.15) has been purified 160-fold from Escherichia coli K12. The molecular weight of the enzyme is 160 000. 2. 2. The enzyme has a broad pH optimum between pH 6 and 8. The initial velocity data obtained follow regular Michaelis-Menten kinetics without any detectable kinetic evidence for subunit interaction. On the basis of kinetic experiments the mechanism of enzyme action is ping-pong, and the first substrate is phosphoenol-pyruvate. The absolute Michaelis constant of the enzyme for both phosphoenol-pyruvate and erythrose 4-phosphate is 1.0 mM. Co2+, at a concentration of 1 mM, increases the enzyme activity about 2-fold. Among other ions tested, Mn2+ slightly activates the enzyme, while other heavy metal ions, such as Cu2+ and Zn2+, are inhibitory. 3. 3. Both elevated and low temperatures reversibly inactivate the enzyme. Phosphoenolpyruvate protects the enzyme against the inactivating effect of heat. The Ks of the enzyme for phosphoenolpyruvate depends on the temperature and decreases with decreasing temperature.

Purification and some properties of rat liver tyrosyl-tRNA synthetase.

Rat liver cytoplasmic tyrosine:tRNA ligase (tyrosine:tRNA ligase, EC 6.1.1.1) was purified by ultracentrifugation, DEAE-cellulose chromatography and repeated phosphocellulose chromatography by more than 1500-fold. The molecular weight of the enzyme was approx. 150 000 as determined by Sephadex G-200 gel filtration. On the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme consisted of two subunits, each of 68 000 daltons. We found the following Km values for the enzyme: 13 micrometer for tyrosine and 1.7 mM for ATP in the ATP:PPi exchange reaction and 13 micrometer for tyrosine, 210 micrometer for ATP and 0.14 micrometer for tRNATyr in the aminoacylation reaction. The rate of tyrosyl-tRNA synthesis was 50-fold lower than that of ATP:PPi exchange. Addition of a saturating amount of tRNA did not affect the rate of ATP:PPi exchange.

Purification and properties of tyrosine-sensitive 3-deoxy-D-arabino-heptolosonate-7-phosphate synthetase of Escherichia coli K12.

1. The tyrosine-sensitive allosteric first enzyme of the aromatic amino acid biosynthetic pathway, 3-deoxy-D-arabinoheptulosonate 7-phosphate synthetase (7-Phospho-2-keto-3-deoxy-D-arabino-heptonate D-erythrose 4-phosphate-lyase (pyruvate phosphorylating), EC 4.1.2.15) has been purified from a mutant strain of Escherichia coli. 2. The enzyme activity was inhibited to 50% at 2-10(-5) M tyrosine and to 90% at 2-10(-4) M tyrosine concentration. At tyrosine concentrations lower than 2-10(-5) M a cooperative interaction between tyrosine binding sites was observed. 3. Co2+ increased the enzyme activity about 2-2.5-fold. The presence of Co2+ ions stabilized the enzyme. EDTA inhibited the enzyme activity, and this inhibition was reversed by Co2+. Tyrosine-sensitive DAHP synthetase seems to be a metal containing enzyme. 4. Kinetic experiments were carried out to study the catalytic action. Contrary to earlier suggestions it is concluded, that the reaction mechanism appears to be more complex--with either the ping-pong or sequential type predominating, depending on conditions.

The specificity of the chemical modification of N6-Δ2-(isopentenyl)adenosine in purified yeast tRNASer

Abstract The specific modification of N6-Δ2-(isopentenyl)adenosine in purified tRNASeryeast by mild treatment with KMnO4 and I2 was studied. N6-Δ2-(isopentenyl)adenosine in tRNASer is specifically modified by iodination, providing us with a suitable method for the quantitative determination of N6-Δ2-(isopentenyl)adenosine in tRNA and for the specific labelling of tRNAs containing this compound. Yeast tRNA was found to contain 114 ± 8 pmol A 260 nm unit of N6-Δ2-(isopentenyl)adenosine and gave three labelled fractions on an RPC-5 column. The product obtained after KMnO4 treatment of tRNASer was not homogeneous. The enzymatic “reisopentenylation” of KMnO4-treated tRNASer resulted in the regeneration of only traces of the original molecule(s). Most of them had been damaged either by the KMnO4 treatment or in the incubation mixture used for “reisopentenylation”.

A simplified assay of enzymes catalyzing ATP-pyrophosphate exchange reactions

Abstract The ATP-pyrophosphate exchange reaction is a widely used method for determination of the activity of enzymes forming enzyme-adenylate intermediates in the course of their catalytic action (1–4). For separation of the labeled ATP formed in the enzyme-catalyzed reaction from the labeled pyrophosphate, the adsorption of ATP on activated Norit is generally used. This method, however, is rather time consuming (considering the extensive washings required for the removal of pyrophosphate from Norit), and its reproducibility is highly dependent on pretreatment of charcoal with acid, Another method of separation applies anion-exchange paper (5). According to our experience, separation on DEAE-cellulose paper (Whatman DE 81) is not sufficient. The present paper describes a simple and reproducible thin-layer chromatographic method for the separation of ATP and pyrophosphate. The system consists of commercial chromatoplates precoated with strong anion-exchange resin (Ionex-25 SB-Ac or Fixion 2X8) and 0.1 m aqueous sodium pyrophosphate, pH 8, as developing solvent.

Contribution to physiological properties of poly(N-vinylpyrrolidone-alt-maleic acid), toxicity and hemagglutination tests

Acute toxicity of the copolymer of poly(N-vinylpyrrolidone-alt-maleic acid) is examined by two routes of parenteral administration. The effect of prolonged daily treatment of animals, the results of hemagglutination tests of the original, and the modified copolymer as polymer drug models are discussed.