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N Esaki

Publications and source records attributed to N Esaki.

At least 91 records · Page 5Linked to original sources
PubMed1994

Studies of the active-site lysyl residue of thermostable aspartate aminotransferase: combination of site-directed mutagenesis and chemical modification.

The gene technological substitution of the cysteinyl residue for the pyridoxal 5'-phosphate-binding lysyl residue (K239) of thermostable aspartate aminotransferase of Bacillus sp. YM-2 led to loss of the activity of the enzyme, which inherently contains no cysteinyl residues. The cysteinyl residue of the mutant enzyme was modified to lysine sulfur analog residues, S-(beta-aminoethyl)cysteine (SAEC), S-(beta-aminopropyl)cysteine (SAPC), and S-(beta-aminoethylthio)cysteine (SATC) with 2-bromoethylamine, 3-bromopropylamine, and 2-mercaptoethylamine, respectively. The modified mutant enzymes showed absorbance at 379 (K239SAEC), 400 (K239SAPC), and 365 nm (K239SATC), whereas the spectrum of the wild-type enzyme exhibited an absorption maximum at 360 nm derived from the internal Schiff base at pH 8.0. The absorption of the modified mutant enzymes at these wavelengths disappeared on reduction with NaCNBH3. This suggests that omega-amino groups of the introduced lysine sulfur analog residue form an internal Schiff base with pyridoxal 5'-phosphate. The modified mutant enzymes showed kcat values of 19.6-0.065% of that of the wild-type enzyme in the overall reaction, and were 10(6)-10(8) times more active than the K239C mutant enzyme. These results suggest that omega-amino groups of the introduced residues of the modified mutant enzyme serve as a catalytic base, and catalysis of the enzyme was affected by the length of the functional side-chain.

Aspartate Aminotransferases↗
PubMed1994

Reconsideration of the essential role of a histidine residue of L-2-halo acid dehalogenase.

His20 of L-2-halo acid dehalogenase from Pseudomonas cepacia MBA4 was suggested to serve as a catalytic base [Biochem. J. (1993) 292, 69-74]. In this study, we substituted Asn or Leu for His19 of L-2-halo acid dehalogenase from Pseudomonas sp. YL, which corresponds to His20 of the P. cepacia enzyme. Although the substrate specificity was affected by the substitution, the susceptibilities of substrate halo acids were not substantially diminished, and the Km and kcat values of the mutant enzymes for L-2-chloropropionate were not significantly different from those of the wild-type enzyme. In addition, the wild-type and mutant enzymes showed the same pH optimum. Accordingly, His19 is not essential for catalysis of L-2-halo acid dehalogenase.

Aspartic Acid↗
PubMed1994

Construction and characterization of chimeric enzyme consisting of an amino-terminal domain of phenylalanine dehydrogenase and a carboxy-terminal domain of leucine dehydrogenase.

Phenylalanine dehydrogenase of Thermoactinomyces intermedius acts preferentially on L-phenylalanine and L-tyrosine, whereas leucine dehydrogenase of Bacillus stearothermophilus acts almost exclusively on L-leucine and some other branched-chain L-amino acids. The two enzymes share a sequence similarity (47%). Aiming at elucidation of the mechanism of substrate recognition by the two amino acid dehydrogenases, we have genetically constructed a chimeric enzyme consisting of an N-terminal domain of phenylalanine dehydrogenase containing the substrate-binding region and a C-terminal domain of leucine dehydrogenase containing the NAD(+)-binding region. The chimeric enzyme purified to homogeneity acted on phenylalanine with a specific activity of 6% of that of the parental phenylalanine dehydrogenase and showed a broad substrate specificity in the oxidative deamination, like phenylalanine dehydrogenase. However, it acted much more effectively than phenylalanine dehydrogenase on isoleucine and valine. Its Km values for L-phenylalanine and L-leucine were similar to those of phenylalanine dehydrogenase. The substrate specificity of the chimeric enzyme in the reductive amination was an admixture of those of the two parent enzymes. These results suggest that the two domains of phenylalanine dehydrogenase and leucine dehydrogenase probably can fold independently. Accordingly, their chimera forms a new active enzyme which consists of their N- and C-terminal domains containing the substrate- and coenzyme-binding regions, respectively. However, the two domains of chimeric enzyme interact and communicate with each other to form a new active site and consistently show the new substrate specificity.

Amino Acid Oxidoreductases↗
PubMed1994

Identification of active site lysyl residues of phenylalanine dehydrogenase by chemical modification with methyl acetyl phosphate combined with site-directed mutagenesis.

A monoanionic acetylation reagent, methyl acetyl phosphate, was used to acetylate lysyl residues of the recombinant thermostable phenylalanine dehydrogenase from Thermoactinomyces intermedius. The enzyme was irreversibly inactivated with the reagent in a time- and dose-dependent manner. Simultaneous addition of substrate and coenzyme markedly protected the enzyme from inactivation. Acetylated lysyl residues presumably occurring at the active site were determined by differential modification; the enzyme was first modified with a cold reagent in the presence of both substrate and coenzyme and, after removal of the added substances by gel filtration, was then labeled with a radioactive reagent. At least 7 lysyl residues per enzyme subunit were radiolabeled by this method. To further specify the lysyl residue(s) whose modification results in inactivation of the enzyme, 5 lysyl residues highly conserved in various amino acid dehydrogenase sequences were replaced with Ala by site-directed mutagenesis. Although all of the single mutant enzymes were inactivated with the reagent as effectively as the wild-type enzyme, a double mutant enzyme in which both Lys-69 and Lys-81 were replaced with Ala was found to be inactivated very slowly. These results suggest that the reagent can acetylate both of these lysyl residues and inactivate the enzyme. Kinetic analyses of the single Lys-69 and Lys-81 mutant enzymes revealed that they are involved in substrate binding and catalysis, respectively, like the corresponding residues in the homologous leucine dehydrogenase.

Amino Acid Oxidoreductases↗
PubMed1994

Purification and characterization of thermostable and nonthermostable 2-haloacid dehalogenases with different stereospecificities from Pseudomonas sp. strain YL.

Two novel hydrolytic dehalogenases, thermostable L-2-haloacid dehalogenase (L-DEX) inducibly synthesized by 2-chloropropionate (2-CPA) and nonthermostable DL-2-haloacid dehalogenase (DL-DEX) induced by 2-chloroacrylate, were purified to homogeneity from Pseudomonas sp. strain YL. DL-DEX consisted of a monomer with a molecular weight of about 36,000 and catalyzed the dehalogenation of L and D isomers of 2-CPA to produce D- and L-lactates, respectively. It acted on 2-haloalkanoic acids with a carbon chain length of 2 to 4. The maximum activity on DL-2-CPA was found at pH 10.5 and 45 degrees C. L-DEX, composed of two subunits with identical molecular weights of 27,000, catalyzes the dehalogenation of L-2-haloalkanoic acids to produce the corresponding D-2-hydroxyalkanoic acids. The enzyme acts not only on short-carbon-chain 2-haloacids such as monochloroacetate and monoiodoacetate in aqueous solution but also on long-carbon-chain 2-haloacids such as 2-bromohexadecanoate in n-heptane. L-DEX is thermostable: it retained its full activity upon heating at 60 degrees C for 30 min. The pH and temperature optima for dehalogenation of L-2-CPA were 9.5 and 65 degrees C, respectively. L-DEX was strongly inhibited by modification of carboxyl groups with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and Woodward reagent K, but DL-DEX was not.

Amino Acid Sequence↗
PubMed1994

Comparative studies of genes encoding thermostable L-2-halo acid dehalogenase from Pseudomonas sp. strain YL, other dehalogenases, and two related hypothetical proteins from Escherichia coli.

We have determined the nucleotide sequence of the gene encoding thermostable L-2-halo acid dehalogenase (L-DEX) from the 2-chloroacrylate-utilizable bacterium Pseudomonas sp. strain YL. The open reading frame consists of 696 nucleotides corresponding to 232 amino acid residues. The protein molecular weight was estimated to be 26,179, which was in good agreement with the subunit molecular weight of the enzyme. The gene was efficiently expressed in the recombinant Escherichia coli cells: the amount of L-DEX corresponds to about 49% of the total soluble proteins. The predicted amino acid sequence showed a high level of similarity to those of L-DEXs from other bacterial strains and haloacetate dehalogenase H-2 from Moraxella sp. strain B (38 to 57% identity) but a very low level of similarity to those of haloacetate dehalogenase H-1 from Moraxella sp. strain B (10%) and haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (12%). By searching the protein amino acid sequence database, we found two E. coli hypothetical proteins similar to the Pseudomonas sp. strain YL L-DEX (21 to 22%).

Amino Acid Sequence↗
PubMed1993

Expression of glr (murI, dga) gene encoding glutamate racemase in Escherichia coli.

The murI (dga) gene of Escherichia coli is required for the biosynthesis of D-glutamate, an essential component of bacterial peptidoglycan (Doublet, P., van Heijetnoort, J., and Mengin-Lecreulx, D. (1992) J. Bacteriol. 174, 5772-5779; Dougherty, T. J., Thanassi, J. A., and Pucci, M. J. (1993) J. Bacteriol. 175, 111-116), but its gene product has not been identified. We found that the amino acid sequence of protein deduced from the nucleotide sequence of the open reading frame of murI gene (ORF1) shows a significant homology with that of glutamate racemase of Pediococcus pentosaceus. The amino acid sequence of glutamate racemase of Lactobacillus fermenti recently reported also shows a homology with the deduced amino acid sequence of ORFI (Gallo, K. A., and Knowles, J. R. (1993) Biochemistry 32, 3981-3990). The murI (dga) gene was ligated into a plasmid, pKK223-3, with a designed ribosome binding site and expressed in E. coli JM109 cells. Glutamate racemase was produced by the transformant cells, whereas the enzyme was not found in the host cells. Accordingly, we newly termed the gene glr, which is more relevant than murI and dga. We partially purified the enzyme to characterize it. The enzyme consists of two identical subunits with a molecular weight of about 31,000 in contrast to the P. pentosaceus enzyme, a monomer protein.

Amino Acid Isomerases↗
PubMed1993

Synthesis and characterization of the selenium analog of glutathione disulfide.

We synthesized the selenium analog of glutathione disulfide by a liquid phase method and named it glutaselenone (i.e., gamma-L-glutamyl-L-selenocysteinylglycine) diselenide. The selenol of selenocysteine was protected by the p-methoxybenzyl group, which was removed by acidolysis with trifluoroacetic acid in the presence of thioanisol. The overall yield of the final product, glutaselenone diselenide, was about 9% based on the starting compound, Se-(p-methoxybenzyl)-L-selenocysteine. Glutaselenone diselenide showed a broad absorption band between 270 and 400 nm and circular dichroism bands around 270 nm (positive) and 330 nm (negative), which were attributable to diselenide bond.

Chromatography, High Pressure Liquid↗
PubMed1993

Site-directed mutagenesis of a hexapeptide segment involved in substrate recognition of phenylalanine dehydrogenase from Thermoactinomyces intermedius.

Phenylalanine dehydrogenase from Thermoactinomyces intermedius and leucine dehydrogenase from Bacillus stearothermophilus show a 59% sequence similarity in their substrate-binding domains, although their substrate specificities are different. We prepared a phenylalanine dehydrogenase mutant enzyme whose inherent hexapeptide segment (124F-V-H-A-A-129R) in the substrate-binding domain was replaced by the corresponding part of leucine dehydrogenase (M-D-I-I-Y-Q) in order to investigate the mechanism of substrate recognition by phenylalanine dehydrogenase. The catalytic efficiencies (kcat/Km) of the mutant enzyme with aliphatic amino acids and aliphatic keto acids as substrates were 0.5 to 2% of those of the wild-type enzyme. In contrast, the efficiencies for L-phenylalanine and phenylpyruvate decreased to 0.008 and 0.035% of those of the wild-type enzyme, respectively. These results suggest that the hexapeptide segment plays an important role in the substrate recognition by phenylalanine dehydrogenase.

Amino Acid Oxidoreductases↗
PubMed1993

A new amino acid racemase with threonine alpha-epimerase activity from Pseudomonas putida: purification and characterization.

We have found that Pseudomonas putida ATCC 17642 cells grown in a medium containing D-threonine as the sole nitrogen source produce an enzyme that catalyzes epimerization of threonine. Proton nuclear magnetic resonance analysis of the enzyme reaction in deuterium oxide clearly showed epimerization from L- to D-allo-threonine and also from D- to L-allo-threonine. This is the first example of an enzyme that was clearly shown to epimerize threonine. The enzyme has been purified to homogeneity, which was shown by polyacrylamide gel electrophoresis. The enzyme has a molecular weight of about 82,000 and consists of two subunits identical in molecular weight (about 41,000). The enzyme contains 1 mol of pyridoxal 5'-phosphate per mol of subunit as a cofactor, and its absorption spectrum exhibits absorption maxima at 280 and 420 nm. The enzyme catalyzes not only epimerization of threonine by stereoconversion at the alpha position but also racemization of various amino acids, except acidic and aromatic amino acids. The enzyme is similar to amino acid racemase with low substrate specificity (EC 5.1.1.10) in enzymological properties but is distinct from it in the action on threonine.

Amino Acids↗
PubMed1993

Synthetic study on selenocystine-containing peptides.

N-9-Fluorenylmethoxycarbonyl-Se-4-methoxybenzylselenocysteine++ + [Fmoc-Sec(MBzl)-OH] was synthesized from selenocystine and successfully applied to Fmoc-based solid-phase peptide synthesis. The stability and the deprotection conditions of the Se-MBzl group were examined. The diselenide bond of a peptide was directly and effectively established between Sec(MBzl) residues by treatment with iodine or the dimethyl sulfoxide-trifluoroacetic acid system. Reduction kinetics of diselenide and disulfide in model peptides by reduced glutathione were also studied comparatively.

Amino Acid Sequence↗
PubMed1993

Thermolabile alanine racemase from a psychotroph, Pseudomonas fluorescens: purification and properties.

A psychotrophic bacterium that produces a thermolabile alanine racemase was isolated from raw milk, and identified as Pseudomonas fluorescens TM5-2. The enzyme was purified to homogeneity from the cell extract, and characterized to be compared with enzymes from mesophiles (Bacillus subtilis and Salmonella typhimurium) and a thermophile (Bacillus stearothermophilus). The enzyme has a molecular weight of about 76,000 and consists of two subunits identical in molecular weight (38,000). The enzyme contains two mol of pyridoxal 5'-phosphate per mol as a coenzyme. The amino acid composition was different from those of other alanine racemases in content of valine. The amino acid sequence of the amino terminal region (from 1Met to 25Gly) had 21-33% homology with those of other alanine racemases. Kinetic parameters of the enzyme were similar to those of other alanine racemases. The enzyme is extremely labile over 30 degrees C, and shows the high catalytic activity even at 0 degrees C; it is thermolabile and psychotrophic.

Alanine Racemase↗
PubMed1993

[Measurement of serum PA values by a newly developed enzyme immunoassay].

Serum prostate-specific antigen (PA) values detected by a newly developed enzyme immunoassay (EIA, MARKIT-M PA) as a successor of MARKIT-F PA, which has been a leading kit in Japan, were evaluated for its role in the diagnosis of cancer of the prostate and follow-up of the patients afflicted with the disease. The system is one-step sandwich type EIA using horseradish peroxidase as a tracer and has 0.50-100 ng/ml of detectable range with small amount of sample volume (25 microliters) and reliable quality control data. Furthermore, serum PA values detected by the assay were almost equivocal to those detected by MARKIT-F PA. Serum PA values in prostate cancer patients (n = 122) were statistically higher than those in normal males (n = 90), urological malignancies other than prostate cancer (n = 48) or benign prostatic hypertrophy (BPH, n = 73). Even in the patients with stage A and B prostate cancer, serum PA values were observed to be statistically higher than those in BPH cases. If 3.6 ng/ml was used, which is normal value in MARKIT-F PA, as a cut-off value and BPH cases as a control, the sensitivity, specificity and efficacy for diagnosis of prostate cancer were 77.9, 91.8 and 83.1%, respectively, which showed the best results during the range examined. Serially determined serum PA values in following up the patients with prostate cancer were confirmed to be highly effective to evaluate treatment responses. These results suggest that MARKIT-M PA is thought to be one of the best tool for determination of serum PA values.

Humans↗
PubMed1992

Properties of aspartate racemase, a pyridoxal 5'-phosphate-independent amino acid racemase.

Aspartate racemase from Streptococcus thermophilus contains no pyridoxal 5'-phosphate or other cofactors such as FAD, NAD+, and metal ions. It was affected by neither carbonyl reagents such as hydroxylamine nor sodium borohydride but was strongly inhibited by iodoacetamide and other thiol reagents. Aspartate, cysteate, and cysteine sulfinate were the only substrates. The Km values for L- and D-aspartate were 35 and 8.7 mM, respectively. The enzyme catalyzed the exchange of alpha-hydrogen of the substrate with the solvent hydrogen. Racemization of L-aspartate in 2H2O showed an overshooting in the optical rotation of aspartate before the substrate was fully racemized. This shows that the removal of alpha-hydrogen of the substrate is at least partially rate-determining. When L- or D-aspartate was incubated with aspartate racemase in tritiated water, tritium was incorporated preferentially into the product enantiomer. The results strongly suggest that aspartate racemase contains two hydrogen acceptors.

Amino Acid Isomerases↗
PubMed1992

Reaction mechanism of glutamate racemase, a pyridoxal phosphate-independent amino acid racemase.

Glutamate racemase of Pediococcus pentosaceus contained no cofactor, and was completely inactivated by a thiol reagent. The role of a cysteine residue in the enzyme reaction was studied by chemical modification. The modification of this cysteine residue resulted in a concomitant loss of activity. DL-Glutamate protected the enzyme from inactivation. The inactivated enzyme was reactivated by addition of dithiothreitol. The racemization in 2H2O showed an overshoot in the optical rotation of glutamate before the substrate was completely racemized. This indicates that the removal of alpha-hydrogen is the rate determining step. During the racemization of D- or L-glutamate in 3H2O, tritium was incorporated preferentially into the product. Glutamate is racemized by the enzyme probably through a two base mechanism.

Amino Acid Isomerases↗
PubMed1992

[Measurement of serum PSA values by DELFIA PSA and its clinical significance on diagnosis and follow-up of prostate cancer patients].

Serum prostate specific antigen (PSA) values detected by DELFIA PSA were evaluated for usefulness in the diagnosis and follow-up of patients with prostate cancer. The system is time-resolved fluoroimmunoassay using europium as a tracer, which has a detectable range of 0.10-500 ng/ml with a small sample volume (25 microliters) and reliable quality control data. Furthermore, serum PSA values detected by the assay were equivocal to those detected by Tandem-R PSA. From the mean +3 S.D. of serum PSA values obtained on 227 normal males, 1.98 ng/ml was decided as an upper normal level. Serum PSA values in benign prostatic hyperplasia (BPH) (n = 69) and prostate cancer (n = 86) patients were statistically higher than those in normal males. However, when 1.98 ng/ml was used as a cut-off value, the false positive rate in BPH cases elevated up to 80%. Therefore, in the differential diagnosis of prostate cancer and BPH, we recommend 11.7 ng/ml (mean + S.D. in BPH cases) as a cut-off value, in which sensitivity is 72.1%, 88.4% are true negative in BPH, and efficacy is 79.4%. Serially determined serum PSA values in following up the patients with prostate cancer were confirmed to be highly effective for diagnosing recurrence and evaluating treatment responses. These findings suggest that DELFIA PSA is a useful tool for determination of serum PSA values.

Adult↗
PubMed1991

Thermostable alanine racemase of Bacillus stearothermophilus. Construction and expression of active fragmentary enzyme.

Limited proteolysis studies on alanine racemase suggested that the enzyme subunit is composed of two domains (Galakatos, N. G., and Walsh, C. T. (1987) Biochemistry 26, 8475-8480). We have constructed a mutant gene that tandemly encodes the two polypeptides of the Bacillus stearothermophilus enzyme subunit cleaved at the position corresponding to the predicted hinge region. The mutant gene product purified was shown to be composed of two sets of the two polypeptide fragments and was immunologically identical to the wild-type enzyme. The mutant enzyme, i.e. the fragmentary alanine racemase, was active in both directions of the racemization of alanine. The maximum velocity (Vmax) was about half that of the wild-type enzyme, and the Km value was about double. Absorption and circular dichroism spectra of the fragmentary enzyme were similar to those of the wild-type enzyme. An attempt was made to separately express in Escherichia coli a single polypeptide corresponding to each domain, but no protein reactive with the antibody against the wild-type alanine racemase was produced. Therefore, it is suggested that the two polypeptide fragments can fold into an active structure only when they are co-translated and that they correspond to structural folding units in the parental polypeptide chain.

Alanine Racemase↗