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Biomedical subjects

N Esaki

Publications and source records attributed to N Esaki.

At least 145 records · Page 8Linked to original sources
PubMed1983

Microbial distribution of selenocysteine lyase.

We studied the distribution of selenocysteine lyase, a novel enzyme catalyzing the conversion of selenocysteine into alanine and H2Se, which we first demonstrated in various mammalian tissues (Esaki et al., J. Biol. Chem. 257:4386-4391, 1982). Enzyme activity was found in various bacteria such as Alcaligenes viscolactis and Pseudomonas alkanolytica. No significant activity was found in yeasts and fungi. Selenocysteine lyases from A. viscolactis and P. alkanolytica acted specifically on L-selenocysteine and required pyridoxal 5'-phosphate as a cofactor.

Alcaligenes↗
PubMed1982

Purification and properties of alpha-amino-epsilon-caprolactam racemase from Achromobacter obae.

We have purified a unique enzyme, alpha-amino-epsilon-caprolactam racemase 945-fold from an extract of Achromobacter obae by Octyl-Sepharose CL-4B and Thiopropyl-Sepharose 6B and some other chromatographies. The purified enzyme was found homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical ultracentrifugation. The enzyme has a monomeric structure with Mr approximately 50000 and a sedimentation coefficient (S20,w) of 4.28 S. The enzyme contains pyridoxal 5'-phosphate as a coenzyme. The pH optimum for the enzyme activity is approximately 9.0. D- and L-alpha-amino-epsilon-caprolactams are the only substrates. The Km values for the D- and L-isomers are, 8 and 6 mM, respectively.

Alcaligenes↗
PubMed1982

Selenocysteine lyase, a novel enzyme that specifically acts on selenocysteine. Mammalian distribution and purification and properties of pig liver enzyme.

We have found a novel enzyme that exclusively decomposes L-selenocysteine into L-alanine and H2Se in various mammalian tissues, and have named it selenocysteine lyase. The enzyme from pig liver has been purified to homogeneity. It has a molecular weight of approximately 85,000, and contains pyridoxal 5'-phosphate as a coenzyme. Its maximum reactivity is at about pH 9.0. Balance studies showed that 1 mol of selenocysteine is converted to equimolar amounts of alanine and H2Se. The following amino acids are insert: L-cysteine, L-serine, L-cysteine sulfinate, selenocysteamine, Se-ethyl-DL-selenocysteine, and L-selenohomocysteine. L-Cysteine (Ki, 1.0 mM) competes with L-selenocysteine (Km, 0.83 mM) to inhibit the enzyme reaction. The enzyme is the first proven enzyme that specifically acts on selenium compounds.

Animals↗
PubMed1982

Bacterial assimilation of D- and L-2-chloropropionates and occurrence of a new dehalogenase.

The occurrence of a new bacterial dehalogenase acting on both the optical isomers of 2-halogenated alkanoic acids was demonstrated. When the haloalkanoic acid-utilizing bacteria were screened in a medium containing DL-2-chloropropionate as a sole carbon source, two types of bacteria were isolated: (1) a few strains utilizing both D- and L-isomers of 2-chloropropionate and (2) strains utilizing only the L-isomer. A dehalogenating enzyme was obtained from the cells of Pseudomonas sp. which is able to utilize both isomers. The crude enzyme catalyzed the dehalogenation of D- and L-2-chloropropionates to yield L- and D-isomers of lactate, respectively. The enzyme showed the same pH optimum and heat inactivation rate for the D- and L-isomers. Apparent Km values for D- and L-2-chloropropionates were 4.5 and 1.0mM, respectively. The enzyme acted specifically on 2-haloalkanoic acids. Activity staining of disc-gels electrophoresed with the crude enzyme preparation showed that the dehalogenation of D- and L-2-chloropropionates, monochloroacetate, dichloroacetate, 2,2-dichloropropionate, and DL-2-chlorobutyrate is due to a single protein.

Cations, Divalent↗
PubMed1982

Purification and properties of a new enzyme, DL-2-haloacid dehalogenase, from Pseudomonas sp.

A new enzyme, DL-2-haloacid dehalogenase, was isolated and purified to homogeneity from the cells of Pseudomonas sp. strain 113. This enzyme catalyzed non-stereospecific dehalogenation of both of the optical isomers of 2-chloropropionate through an SN2 type of reaction; L- and D-lactates were formed from D- and L-2-chloropropionates, respectively. The enzyme acted on 2-halogenated aliphatic carboxylic acids whose carbon chain lengths were less than five. It also dehalogenated trichloroacetate to form oxalate and showed maximum activity at pH 9.5. The Michaelis constants for substrates were as follows: 5.0 mM for monochloroacetate, 1.1 mM for L-2-chloropropionate, and 4.8 mM for D-2-chloropropionate. DL-2-Haloacid dehalogenase was inhibited by HgCl2, ZnSO4, and MnSO4, but was not affected by thiol reagents, such as p-chloromercuribenzoate and iodoacetamide. This enzyme had a molecular weight of about 68,000 and appeared to be composed of two subunits identical in molecular weight.

Acetates↗
PubMed1981

Mechanistic studies on reactions of bacterial methionine gamma-lyase with olefinic amino acids.

Methionine gamma-lyase (EC 4.4.1.11), which catalyzes the formation of methanethiol, alpha-ketobutyrate, and ammonia from L-methionine (eq 1), promotes the oxidative deamination of several four- and five-carbon olefinic amino acids (1-5). With the exception of vinylglycine (1), the Vmax rates of keto acid formation from the unsaturated substrate analogues are substantially lower than that for processing of methionine to alpha-ketobutyrate; vinylglycine is deaminated to ketobutyrate and ammonia with a Vmax twice that for L-methionine turnover. L-Allylglycine, L-2-amino-3-trans-pentenoate, and L-2-amino-3-cis-pentenoate (2, 4, 5) are all converted to 2-keto-pentanoic acid (alpha-ketovalerate). L-2-Amino-3-cis-pentenoate (5) is also a time-dependent, irreversible inactivator of the enzyme. None of the other substrate analogues tested appears to inactivate the enzyme. Spectral analysis of the enzymatic reaction with cis isomer 5 reveals the formation of a high-wavelength chromophore (lambda max = 550 nm ) which implies that a beta, gamma-unsaturated pyridoxal p-quinoid (VI) accumulates. No such absorbing species appears to form during the reaction of trans isomer 4 with methionine gamma-lyase. But a 550-nm chromophore develops when both 4 and 5 are reacted with Al(NO3)3 and pyridoxal methochloride in methanolic KOH. It would appear that the geometry of the protein and the olefinic amino acid as an intermediate enzyme-substrate adduct controls the kinetics of reaction, such that azaallylic isomerization becomes selectively rate determining for reaction with 5. When this isomerization is slow, an accumulating Michael-type acceptor (VI) could lead to the observed irreversible inactivation of the enzyme.

Alkenes↗
PubMed1981

Enzymatic synthesis of selenocysteine in rat liver.

We have investigated selenocysteine (2-amino-3-hydroselenopropionic acid) synthesis with cystathionine beta-synthase (EC 4.2.1.22) and cystathionine gamma-lyase (EC 4.4.1.1) of rat liver. When selenohomocysteine and serine were incubated with cystathionine beta-synthase, selenocystathionine was formed at a rate of 69% of that of cystathionine synthesis. Cystathionine gamma-lyase catalyzed alpha, gamma elimination of selenocystathionine to yield alpha-ketobutyrate, selenocysteine, and NH3. The reaction rate was about 3 times higher than that of cystathionine elimination. Cystathionine beta-synthase, however, did not catalyze direct formation of selenocysteine from serine and H2Se. Thus, selenocysteine is synthesized from selenohomocysteine and cystathionine beta-synthase and cystathionine gamma-lyase reactions. We confirmed this synthetic pathway also with a mixture of both enzymes and with a homogenate of rat liver.

Animals↗
PubMed1979

Suicide inactivation of bacterial cystathionine gamma-synthase and methionine gamma-lyase during processing of L-propargylglycine.

L-Propargylglycine, a naturally occurring gamma, delta-acetylenic alpha-amino acid, induces mechanism-based inactivation of two pyridoxal phosphate dependent enzymes of methionine metabolism: (1) cystathionine gamma-synthease, which catalyzes a gamma-replacement reaction in methionine biosynthesis, and (2) methionine gamma-lyase, which catalyzes a gamma-elimination reaction in methionine breakdown. Biphasic pseudo-first-order inactivation kinetics were observed for both enzymes. Complete inactivation is achieved with a minimum molar ratio ([propargylglycine]/[enzyme monomer]) of 4:1 for cystathionine gamma-synthase and of 8:1 for methionine gamma-lyase, consistent with a small number of turnovers per inactivation event. Partitioning ratios were determined directly from observed primary kinetic isotope effects. [alpha-2H]Propargylglycine displays kH/kD values of about 3 on inactivation half-times. [alpha-3H]-Propargylglycine gives release of tritium to solvent nominally stoichiometric with inactivation but, on correction for the calculated tritium isotope discrimination, partition ratios of four and six turnovers per monomer inactivated are indicated for cystathionine gamma-synthase and methionine gamma-lyase, respectively. The inactivation stoichiometry, using [alpha-14C]-propargylglycine, is four labels per tetramer of cystathionine gamma-synthase but usually only two labels per tetramer of methionine gamma-lyase (half-of-the-sites reactivity). Two-dimensional urea isoelectrofocusing/NaDodSO4 electrophoresis suggests (1) that both native enzymes are alpha 2 beta 2 tetramers where the subunits are distinguishable by charge but not by size and (2) that, while each subunit of a cystathionine gamma-synthase tetramer becomes modified by propargylglycine, only one alpha and one beta subunit may be labeled in an inactive alpha 2 beta 2 tetramer of methionine gamma-lyase. Steady-state spectroscopic analyses during inactivation indicated that modified cystathionine gamma-synthase may reprotonate C2 of the enzyme--inactivator adduct, so that the cofactor is still in the pyridoxaldimine oxidation state. Fully inactivated methionine gamma-lyase has lambda max values at 460 and 495 nm, which may represent conjugated pyridoximine paraquinoid that does not reprotonate at C2 of the bound adduct. Either species could arise from Michael-type addition of an enzymic nucleophile to an electrophilic 3,4-allenic paraquinoid intermediate, generated initially by propargylic rearrangement upon a 4,5-acetylenic pyridoximine structure, as originally proposed for propargylglycine inactivation of gamma-cystathionase [Abeles, R., & Walsh, C. (1973) J. Am. Chem. Soc. 95, 6124]. It is reasonable that cystathionine gamma-synthase is the major in vivo target for this natural acetylenic toxin, the growth-inhibitory effects of which are reversed by methionine.

Alkynes↗
PubMed1979

Catalytic action of L-methionine gamma-lyase on selenomethionine and selenols.

We examined the catalytic action of L-methionine gamma-lyase (EC 4.4.1.11) on selenomethionine (2-amino-4-(methylseleno)butyric acid), methaneselenol, l-hexaneselenol, and benzeneselenol. The enzyme catalyzes alpha, gamma-elimination of selenomethionine to yield alpha-letobutyrate, ammonia, and methaneselenol, and also its gamma-replacement reaction with various thiols to produce S-substituted homocysteines. Selenomethionine is an even better substrate than methionine in alpha, gamma-elimination but is less effective in gamma-replacement. In addition, L-methionine gamma-lyase catalyzes gamma-replacement reaction of methionine and its derivatives with selenols to form the corresponding Se-substituted selenohomocysteines, although selenols are less efficient substituent donors than thiols. This is the first proven mechanism for the incorporation of selenium atom into amino acids.

Benzene Derivatives↗
PubMed1977

Properties of L-methionine gamma-lyase from Pseudomonas ovalis.

The distribution of bacterial L-methionine gamma-lyase (L-methionine methanethiollyase (deaminating) (EC 4.4.1.11) was investigated, and Pseudomonas ovalis (IFO 3738) was found to have the highest activity of enzyme, which was inducibly formed by addition of L-methionine to the medium. L-Methionine gamma-lyase, purified to homogeneity from Ps. ovalis, has a molecular weight of about 173 000 and consists of nonidentical subunits (mol wt: 40 000 and 48 000). The enzyme exhibits absorption maxima at 278 and 420 nm, and a shoulder around 330 nm, which are independent of the pH (6.0 to 10.0), and contains 4 mol of pyridoxal 5'-phosphate per mol of the enzyme. The formyl group of pyridoxal 5'-phosphate is bound in an aldimine linkage to the epsilon-amino group of lysine residues of the protein. The holoenzyme is resolved to the apoenzyme by incubation with hydroxylamine, and reconstituted by addition of pyridoxal 5'-phosphate. The enzyme activity is significantly affected by both carbonyl and sulfhydryl reagents. L-Methionine gamma-lyase catalyzes alpha,gamma- and alpha,beta-elimination reactions of, in addition to L-methionine, several derivatives of L-methionine and L-cysteine, e.g., L-ethionine, DL-methionine sulfone, L-homocysteine, and S-methyl-L-cysteine. The enzyme catalyzes also gamma-replacement reactions of the thiomethyl group of methionine with various alkanethiols (C2-C7), arylthio alcohols (benzenethiol and beta-naphthalenethiol) and the derivatives of ethanethiol (2-mercaptoethanol and cysteamine) to yield the corresponding S-substituted homocysteine. The thiomethyl group of S-methyl-L-cysteine also is replaced by ethanethiol to form S-ethyl-L-cysteine.

Carbon-Sulfur Lyases↗