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N Esaki

Publications and source records attributed to N Esaki.

At least 127 records · Page 7Linked to original sources
PubMed1988

Gene cloning and sequence determination of leucine dehydrogenase from Bacillus stearothermophilus and structural comparison with other NAD(P)+-dependent dehydrogenases.

The gene for leucine dehydrogenase (EC 1.4.1.9) from Bacillus stearothermophilus was cloned and expressed in Escherichia coli. The selection for the cloned gene was based upon activity staining of the replica printed E. coli cells. A transformant showing high leucine dehydrogenase activity was found to carry an about 9 kilobase pair plasmid, which contained 4.6 kilobase pairs of B. stearothermophilus DNA. The nucleotide sequence including the 1287 base pair coding region of the leucine dehydrogenase gene was determined by the dideoxy chain termination method. The translated amino acid sequence was confirmed by automated Edman degradation of several peptide fragments produced from the purified enzyme by trypsin digestion. The polypeptide contained 429 amino acid residues corresponding to the subunit (Mr 49,000) of the hexameric enzyme. Comparison of the amino acid sequence of leucine dehydrogenase with those of other pyridine nucleotide dependent oxidoreductases registered in a protein data bank revealed significant sequence similarity, particularly between leucine and glutamate dehydrogenases, in the regions containing the coenzyme binding domain and certain specific residues with catalytic importance.

Amino Acid Oxidoreductases↗
PubMed1988

Specific labeling of the essential cysteine residue of L-methionine gamma-lyase with a cofactor analogue, N-(bromoacetyl)pyridoxamine phosphate.

L-Methionine gamma-lyase from Pseudomonas putida is composed of four identical polypeptide chains and contains four cysteinyl residues per subunit. We have found one of them catalytically essential by its specific cyanylation with 2-nitro-5-thiocyanobenzoic acid. We have shown its essentiality also with N-(bromoacetyl)pyridoxamine 5'-phosphate (BAPMP), which is a cofactor analogue and also an affinity-labeling agent. The kinetic data show that the apoenzyme forms a binary complex with BAPMP prior to covalent binding. The stoichiometry of inactivation was 1 mol of BAPMP per subunit. We have shown that the cysteine residue modified with BAPMP is identical with that labeled specifically with [14C]iodoacetic acid. The amino acid sequences of the peptides containing the essential cysteine residue and the lysine residue to which pyridoxal 5'-phosphate is bound were determined by automated Edman degradation.

Affinity Labels↗
PubMed1988

Chemical synthesis and expression of copper metallothionein gene of Neurospora crassa.

The gene coding for the Neurospora crassa copper metallothionein (MT) was synthesized and inserted in the lacZ' gene of pUC18 plasmid to give the same translational reading frame as the latter gene. The MT-beta-galactosidase fused gene was expressed in Escherichia coli to produce a fused protein in which the amino and carboxy termini of MT are linked to the beta-galactosidase through methionine residues. An MT derivative containing an extra homoserine residue at the carboxy terminus was prepared by cyanogen bromide cleavage of the fused protein followed by a reverse-phase HPLC separation. The spectral features of the MT derivative and its copper complex were similar to those of the corresponding native MTs.

Amino Acid Sequence↗
PubMed1988

Purification and characterization of Clostridium sticklandii D-selenocystine alpha, beta-lyase.

We have found a novel enzyme that decomposes D-selenocystine into pyruvate, ammonia, and elemental selenium in extracts of Clostridium sticklandii and C. sporogenes. The enzyme of C. sticklandii has been purified to homogeneity. It has a molecular weight of 74,000 and consists of two subunits identical in molecular weight (35,000). Pyridoxal 5'-phosphate is required as a cofactor. In addition to D-selenocystine, D-cystine, D-lanthionine, meso-lanthionine, and D-cysteine serve as substrates. However, D-selenocysteine, D-serine, DL-selenohomocystine, and L-amino acids are inert. The enzyme also catalyzes the beta-replacement reaction between D-selenocystine and a thiol to produce S-substituted D-cysteine. L-Selenohomocysteine also can serve as a substituent donor in the beta-replacement reaction to yield selenocystathionine.

Chemical Phenomena↗
PubMed1987

Selenocysteine.

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Chromatography↗
PubMed1986

Biosynthetic alanine racemase of Salmonella typhimurium: purification and characterization of the enzyme encoded by the alr gene.

An alanine racemase, encoded by the alr (dal) gene and believed to be the biosynthetic source of D-alanine for cell wall formation, was purified to homogeneity from an overproducing strain of Salmonella typhimurium (dadB), and the enzymological properties of this enzyme were compared with those of the dadB alanine racemase that functions in the catabolism of L-alanine [Wasserman, S. A., Daub, E., Grisafi, P., Botstein, D., & Walsh, C. T. (1984) Biochemistry 23, 5182]. The alr-encoded enzyme has a monomeric structure with a molecular weight of about 40 000. One mole of pyridoxal 5'-phosphate is bound per mole of enzyme, which is essential for catalytic activity of the enzyme. After the internal Schiff base with pyridoxal 5'-phosphate was reduced with NaB3H4, followed by carboxamidomethylation and tryptic digestion of the enzyme, the amino acid sequence of the pyridoxal 5'-phosphate binding peptide was determined. The sequence of 10 amino acid residues around the lysine residue, to which pyridoxal 5'-phosphate is bound, was identical with that of the dadB racemase. No homology was found in the amino-terminal amino acid sequence between the two enzymes. The enzyme was inactivated with D- and L-beta-fluoroalanine, D- and L-beta-chloroalanine, and D-O-acetylserine in a mechanism-based fashion with a common partition ratio of about 150. The enzyme was labeled with an equimolar amount of [14C]-D-beta-chloroalanine. The inactivator-pyridoxal 5'-phosphate adduct was isolated and shown to be the same structure formed in the dadB racemase inactivation [Roise, D., Soda, K., Yagi, T., & Walsh, C. (1984) Biochemistry 23, 5195].

Alanine Racemase↗
PubMed1986

Mechanism of alpha-amino-epsilon-caprolactam racemase reaction.

alpha-Amino-epsilon-caprolactam racemase catalyzes the exchange of the alpha-hydrogen of the substrate with deuterium during racemization in deuterium oxide. The rate of the hydrogen exchange measured by 1H NMR is lower than that of racemization in deuterium oxide for both the enantiomers. Both the enantiomers of alpha-amino-epsilon-caprolactam show an overshoot of the optical rotation during the enzymatic racemization in deuterium oxide (but not in water). This phenomenon may be attributable to a primary deuterium isotope effect at the alpha-position: alpha-deuterium isotope effects of 3.6 and 2.0 were observed for the racemization of the D and L enantiomers of alpha-amino-epsilon-caprolactam, respectively. Results of tritium-labeling experiments showed that the enzyme catalyzes both retention and inversion of configuration of the substrate with a similar probability in each turnover. Conversion of [alpha-2H]-D-alpha-amino-epsilon-caprolactam in water and unlabeled D-alpha-amino-epsilon-caprolactam in deuterium oxide into the L isomer under nearly single turnover conditions with the enzyme showed significant internal return of the alpha-hydrogen. These results support a single base mechanism for the racemization reaction catalyzed by the enzyme.

Alcaligenes↗
PubMed1985

Synthesis of L-selenodjenkolate and its degradation with methionine gamma-lyase.

A convenient method for the synthesis of a new selenium-containing amino acid, L-selenodjenkolate (3,3'-methylenediselenobis(2-aminopropionic acid)), is described. The starting material, selenocystine, was found to be prepared easily from commercially available beta-chloro-L-alanine, elemental selenium, and sodium borohydride. These synthetic procedures are useful for the preparation of the isotope-labeled compounds. The physiochemical properties of selenodjenkolate thus prepared are reported. This amino acid undergoes alpha, beta-elimination to produce pyruvate, formaldehyde, ammonia, and selenium by bacterial methionine gamma-lyase under aerobic conditions: the Vmax and Km values were determined to be 0.5 mumol/min/mg and 2.3 mM, respectively.

Alanine↗
PubMed1985

1H NMR studies of substrate hydrogen exchange reactions catalyzed by L-methionine gamma-lyase.

Hydrogen exchange reactions of various L-amino acids catalyzed by L-methionine gamma-lyase (EC 4.4.1.11) have been studied. The enzyme catalyzes the rapid exchange of the alpha- and beta-hydrogens of L-methionine and S-methyl-L-cysteine with deuterium from the solvent. The rate of alpha-hydrogen exchange was about 40 times faster than that of the enzymatic elimination reaction of the sulfur-containing amino acids. The enzyme also catalyzes the exchange reaction of alpha- and beta-hydrogens of the following straight-chain L-amino acids which are not susceptible to elimination: norleucine, norvaline, alpha-aminobutyrate, and alanine. The exchange rates of the alpha-hydrogen and the total beta-hydrogens of L-alanine and L-alpha-aminobutyrate with deuterium followed first-order kinetics. For L-norvaline, L-norleucine, S-methyl-L-cysteine, and L-methionine, the rate of alpha-hydrogen exchange followed first-order kinetics, but the rate of total beta-hydrogen exchange decreased due to a primary isotope effect at the alpha-position. One beta-hydrogen of S-methyl-L-cysteine was exchanged faster than the other, although both the beta-hydrogens were exchanged completely with deuterium ultimately. L-Phenylalanine and L-tryptophan slowly underwent alpha-hydrogen exchange. The pro-R hydrogen of glycine was deuterated stereospecifically. None of the following amino acids were susceptible to the enzymatic hydrogen exchange: D isomers of the above amino acids, branched chain L-amino acids, acidic L-amino acids, and basic L-amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon-Sulfur Lyases↗
PubMed1985

Mechanism of reactions catalyzed by selenocysteine beta-lyase.

The reaction mechanism of selenocystine beta-lyase has been studied and it was found that elemental selenium is released enzymatically from selenocysteine, and reduced to H2Se nonenzymatically with dithiothreitol or some other reductants that are added to prepare selenocysteine from selenocystine in the anaerobic reaction system. 1H and 13C NMR spectra of L-alanine formed in 2H2O have shown that an equimolar amount of [beta-2H1]- and [beta-2H2]alanines are produced. The deuterium isotope effect at the alpha position was observed; kH/kD = 2.4. These results indicated that the alpha hydrogen of selenocysteine was removed by a base at the active site, and was incorporated into the alpha position of alanine, a product, without exchange of a solvent deuterium. When the enzyme was incubated with L-selenocysteine in the absence of added pyridoxal 5'-phosphate, the activity decreased with prolonged incubation time. However, the activity was recovered by addition of 5'-phosphate. The spectrophotometric study showed that the inactivated enzyme was the apo form. The apoenzyme was activated by a combination of pyridoxamine 5'-phosphate and various alpha-keto acids such as alpha-ketoglutarate and pyruvate. Thus, the enzyme is inactivated through transamination between selenocysteine and the bound pyridoxal 5'-phosphate to produce pyridoxamine 5'-phosphate and a keto acid derived from selenocysteine. The pyridoxal enzyme, an active form, is regenerated by addition of alpha-keto acids. This regulatory mechanism is analogous to those of aspartate beta-decarboxylase [EC 4.1.1.12], arginine racemase [EC 5.1.1.9], and kynureninase [EC 3.7.1.3] [K. Soda and K. Tanizawa (1979) Adv. Enzymol. 49, 1].

Alanine↗
PubMed1985

Purification and characterization of selenocysteine beta-lyase from Citrobacter freundii.

The purification and characterization of bacterial selenocysteine beta-lyase, an enzyme which specifically catalyzes the cleavage of L-selenocysteine to L-alanine and Se0, are presented. The enzyme, purified to near homogeneity from Citrobacter freundii, is monomeric with a molecular weight of ca. 64,000 and contains 1 mol of pyridoxal 5'-phosphate as a cofactor per mol of enzyme. L-Selenocysteine is the sole substrate (Km, 0.95 mM). L-Cysteine is a competitive inhibitor of the enzyme (Ki, 0.65 mM). The enzyme also catalyzes the alpha, beta elimination of beta-chloro-L-alanine to form NH3, pyruvate, and Cl- and is irreversibly inactivated during the reaction. The physicochemical properties, e.g., amino acid composition and subunit structure, of the bacterial enzyme are fairly different from those of the pig liver enzyme (Esaki et al., J. Biol. Chem. 257:4386-4391, 1982). However, the catalytic properties of both enzymes, e.g., substrate specificity and inactivation by the substrate or a mechanism-based inactivator, beta-chloro-L-alanine, are very similar.

Amino Acids↗
PubMed1984

Purification of bacterial L-methionine gamma-lyase.

A rapid procedure for the purification of L-methionine gamma-lyase from Pseudomonas putida ICR 3460 by DEAE- TOYOPEARL 650M and DEAE-Sephadex A-50 column chromatography is presented. The enzyme was purified with an average yield of 75% and showed about 10-fold higher specific activity than the enzyme from P. putida (= P. ovalis) IFO 3738 reported previously (H. Tanaka, N. Esaki , and K. Soda (1976) FEBS Lett. 66, 307-311). The present enzyme has a molecular weight of about 172,000 and consists of four subunits with identical molecular weights (43,000). It shows the typical absorption spectrum of pyridoxal enzyme with maxima at 278 and 420 nm, and contains 4 mol of pyridoxal 5'-phosphate per mole of enzyme. The enzyme has a multicatalytic function similar to the enzyme of P. putida IFO 3738 (K. Soda, H. Tanaka, and N. Esaki (1983) Trends Biochem. Sci. 8, 214-217).

Carbon-Sulfur Lyases↗