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K Soda

Publications and source records attributed to K Soda.

At least 271 records · Page 15Linked to original sources
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

Oxidation of anionic nitroalkanes by flavoenzymes, and participation of superoxide anion in the catalysis.

The reactivities of anionic nitroalkanes with 2-nitropropane dioxygenase of Hansenula mrakii, glucose oxidase of Aspergillus niger, and mammalian D-amino acid oxidase have been compared kinetically. 2-Nitropropane dioxygenase is 1200 and 4800 times more active with anionic 2-nitropropane than D-amino acid oxidase and glucose oxidase, respectively. The apparent Km values for anionic 2-nitropropane are as follows: 2-nitropropane dioxygenase, 1.61 mM; glucose oxidase, 16.7 mM; and D-amino acid oxidase, 11.1 mM. Anionic 2-nitropropane undergoes an oxygenase reaction with 2-nitropropane dioxygenase and glucose oxidase, and an oxidase reaction with D-amino acid oxidase. In contrast, anionic nitroethane is oxidized through an oxygenase reaction by 2-nitropropane dioxygenase, and through an oxidase reaction by glucose oxidase. All nitroalkane oxidations by these three flavoenzymes are inhibited by Cu and Zn-superoxide dismutase of bovine blood, Mn-superoxide dismutases of bacilli, Fe-superoxide dismutase of Serratia marcescens, and other O2-. scavengers such as cytochrome c and NADH, but are not affected by hydroxyl radical scavengers such as mannitol. None of the O2-. scavengers tested affected the inherent substrate oxidation by glucose oxidase and D-amino acid oxidase. Furthermore, the generation of O2-. in the oxidation of anionic 2-nitropropane by 2-nitropropane dioxygenase was revealed by ESR spectroscopy. The ESR spectrum of anionic 2-nitropropane plus 2-nitropropane dioxygenase shows signals at g1 = 2.007 and g11 = 2.051, which are characteristic of O2-.. The O2-. generated is a catalytically essential intermediate in the oxidation of anionic nitroalkanes by the enzymes.

Alkanes↗
PubMed1984

Inactivation of the Pseudomonas striata broad specificity amino acid racemase by D and L isomers of beta-substituted alanines: kinetics, stoichiometry, active site peptide, and mechanistic studies.

Mechanism-based inactivators were used to probe the active site of the broad specificity amino acid racemase from Pseudomonas striata. Kinetic parameters for the inactivation of the racemase with both stereoisomers of beta-fluoroalanine, beta-chloroalanine, and O-acetylserine were determined. By use of 14C-labeled O-acetylserines, the stoichiometry of inactivator binding was found to be one inactivator bound per enzyme subunit. The PLP-dependent enzyme contains one coenzyme per subunit, and after NaB3H4 reduction of the PLP-imine bond, followed by trypsin digestion of the protein, the amino acid sequence of the PLP-binding peptide was determined. Trypsin digestion of the enzyme labeled with either L or D isomer of O-acetylserine and sequencing of the labeled peptide revealed that the inactivators bind to the same lysine residue which binds PLP in native enzyme. The characterization of a PLP adduct released from inactivated enzyme under some conditions is also described. Implications of the formation of this compound with respect to the overall reaction mechanism of inactivation are discussed.

Alanine↗
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↗
PubMed1984

Dynamic light-scattering studies on thermal motions of native DNAs in solution.

The basic character of dynamic light-scattering properties of native DNA was investigated on two DNA samples. The degree of non-single exponentiality of photocount correlation functions, C(t), and its dependence on K are quantitatively characterized by two methods. The spectral linewidth, Ts, determined from C(t) exhibits a K dependence near to but significantly different from the prediction for Rouse-Zimm (RZ) chains by Dubois-Violette and De Gennes: It is inferred from data on lambda-DNA that the exponent in the K dependence of the spectral linewidth for native DNA takes a value larger than 3 in the K region corresponding to the 'K3' region for RZ chains. These results are in good agreement with the prediction from the dynamic theory of semiflexible chains presented by one of us (K.S.). The apparent diffusion coefficients are fairly insensitive to DNA concentration and ionic strength at large K. On the other hand, it is indicated that the stiffness of native DNA may vary with temperature even in a temperature range substantially lower than that of melting.

Bacteriocin Plasmids↗
PubMed1984

D-glucosaminate dehydratase: spectrometric properties of the enzyme-bound pyridoxal 5'-phosphate.

The holoenzyme of D-glucosaminate dehydratase [EC 4.2.1.26] from Agrobacterium radiobacter showed absorption peaks at 280 and 415 nm with a shoulder in the region of 320 to 330 nm. The treatment of the enzyme with hydroxylamine followed by dialysis led to disappearance of both the absorption peak at 415 nm and the shoulder, giving the apoenzyme. The fluorescence excitation maximum of the holoenzyme was at 320 nm with a shoulder at 420 nm (emission at 510 nm), and the emission maxima were at 420 nm with a shoulder at 370 nm (excitation at 320 nm) and at 510 nm (excitation at 420 nm). The holoenzyme showed a negative circular dichroic band at 418 nm and a positive shoulder at around 320 nm. Reduction of the holoenzyme with sodium borohydride caused a loss of the absorption peak at 415 nm with a concomitant increase of 325 nm absorbance and an irreversible loss of the activity. The occurrence of epsilon-N-pyridoxyllysine in the acid hydrolysate of the reduced enzyme showed that D-glucosaminate dehydratase contains a catalytically essential lysine residue whose epsilon-amino group binds the 4-formyl group of pyridoxal 5'-phosphate to form a Schiff base. The plots of absorption of the apoenzyme against the amount of pyridoxal 5'-phosphate added showed that four and two molar equivalents of the cofactor bind to the apoenzyme and subunit, respectively. The biphasic nature of the spectrometric titration curve of the apoenzyme with pyridoxal 5'-phosphate and the two Km values obtained for the cofactor suggest the occurrence of two distinct types of binding sites for pyridoxal 5'-phosphate in the enzyme.

Circular Dichroism↗
PubMed1984

Stereochemistry of ornithine decarboxylase reaction.

To determine the steric course of the reaction of bacterial ornithine decarboxylase [EC 4.1.1.17], we have carried out the decarboxylation of L-ornithine in 2H2O and that of DL-[2-2H]ornithine in H2O, and obtained putrescine bearing a single deuterium atom in the C-1 position. The stereochemistry of [1-2H]putrescine was established by conversion to 1-(2-pyrrolidinyl)-2-propanone with acetoacetate and the pro-S hydrogen-specific diamine oxidase from pea seedlings. Analysis of deuterium content by gas chromatography-mass spectrometry showed that the deuterium label was fully retained during the conversion of [1-2H]putrescine produced by the decarboxylation of L-ornithine in 2H2O to 1-(2-pyrrolidinyl)-2-propanone, in contrast with the considerable loss of label from [1-2H]putrescine which was produced by the decarboxylation of DL-[2-2H]ornithine in H2O. The extent of loss of the deuterium label was in good agreement with the estimated value based on the isotope effect in the diamine oxidase reaction. These results indicate that the introduced deuterium (or hydrogen) is in the pro-R position at C-1 of putrescine, and consequently the ornithine decarboxylase reaction proceeds with retention of configuration.

Chemical Phenomena↗
PubMed1984

The effect of carboxylates and halides on L-lysine 6-aminotransferase-catalyzed reactions.

L-Lysine:2-oxoglutarate 6-aminotransferase catalyzes very slow transamination between L-alanine and 2-oxoglutarate. A high concentration of anions such as formate, acetate and halides greatly accelerated this transamination without affecting the affinity of the enzyme for L-alanine. In contrast, the anions strongly inhibited the normal L-lysine 6-transamination in a competitive manner with L-lysine and in a non-competitive manner with 2-oxoglutarate. This result suggests that the enzyme has an anion binding site which normally binds the carboxyl group of L-lysine. The binding of halides or carboxylates to this site probably induces a conformational change of the enzyme, and results in the inhibition of L-lysine 6-transamination, and in the stimulation of L-alanine transamination. Treatment of the enzyme with an arginine-specific dicarbonyl reagent, phenylglyoxal, led to a loss of the enzyme activity for L-lysine. The activity for L-alanine was not affected, but the stimulating effect of anions on L-alanine transamination was impaired. Thus, it is suggested that an arginine residue(s) plays an important role in the anion binding site.

Alanine↗
PubMed1984

Purification and properties of amino acid racemase from Aeromonas punctata subsp. caviae.

An amino acid racemase, which occurs in the cytoplasmic fraction of Aeromonas punctata subsp. caviae, has been purified to homogeneity by the criteria of electrophoresis and ultracentrifugation. The enzyme has a molecular weight of about 80,000 and consists of two subunits identical in molecular weight (about 40,000). The enzyme contains 2 mol of pyridoxal 5'-phosphate per mol of enzyme, and exhibits absorption maxima at 280 nm and 420 nm. The holoenzyme is resolved by dialysis against hydroxylamine to yield the inactive apoenzyme, which is reconstituted by the addition of pyridoxal 5'-phosphate to recover the full activity. The enzyme catalyzes racemization of a number of amino acids, e.g. lysine, ornithine, ethionine, arginine, glutamine, and methionine. The Michaelis constants were determined: 1 mM for L-lysine; 0.9 mM for D-lysine; 0.9 mM for L-ornithine; 1 mM for L-arginine; and 2.6 microM for pyridoxal 5'-phosphate. This enzyme is similar in enzymological properties to the racemase of Pseudomonas putida, but is distinct from it in immunochemical properties.

Aeromonas↗
PubMed1983

Stereochemistry of an alpha, beta-elimination reaction by D-glucosaminate dehydratase.

The proton NMR analysis of D-glucosaminate dehydratase reaction in D2O revealed the incorporation of a deuterium atom at C-3 carbon of the product, 2-keto-3-deoxy-D-gluconate. Based on the chemical shift of C-3 proton of the product and the coupling constant characteristic for the C-3 and C-4 axial-axial coupling in the 2C5 pyranose conformation, the deuterium is in the pro-S position. Thus, the dehydration of D-glucosaminate by the enzyme proceeds in a retention mode at C-3 carbon. Kinetic parameters show that the rate-determining step is the abstraction of alpha-proton from the substrate.

Deuterium↗
PubMed1983

A spectrophotometric method for the determination of aminopeptidase activity with leucine dehydrogenase.

L-Leucine dehydrogenase purified from Bacillus megaterium and Bacillus sphearicus was used for the determination of serum aminopeptidase activity with L-leucinamide as a substrate. L-Leucine produced by aminopeptidase was determined by measurement of the increase in absorbance at 340 nm caused by the formation of NADH. This method is useful for the kinetic studies of the aminopeptidase and the enzyme assay of a large number of samples. The serum aminopeptidase can be characterized to give some valuable information in clinical diagnosis by comparison of the results obtained by the present method with those by the conventional method with L-leucyl-p-nitroanilide as a substrate.

Amino Acid Oxidoreductases↗
PubMed1983

An enzymic cycling method for the determination of free fatty acids with acyl-CoA synthetase and acyl-CoA hydrolase.

A method for measuring free fatty acids by enzymic cycling is described. Free fatty acids are converted to acyl-CoAs by acyl-CoA synthetase, then the acyl-CoAs are hydrolyzed back to the free fatty acids by acyl-CoA hydrolase in a cyclic fashion. The amounts of AMP produced during this cyclic reaction are determined from the absorbance at 340 nm in the presence of AMP deaminase and glutamate dehydrogenase. This method is sensitive to as low as 0.1 nmol of free fatty acids, and the standard curve is linear up to 1.0 nmol. This method shows a broad specificity for long-chain fatty acids (C12--C20) and the recoveries of fatty acids added to bacterial cell-free extracts are more than 90%.

AMP Deaminase↗