Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.47 (cysteine synthase)
625 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A technique based on resistance to azaserine was used to isolate mutants lacking O-acetylserine sulfhydrylase B, one of two enzymes in Salmonella typhimurium capable of synthesizing L-cysteine from O-acetyl-L-serine and sulfide. The mutant locus responsible for this defect has been designated cysM, and genetic mapping suggests that cysM is very close to and perhaps contiguous with cysA. Strains lacking either O-acetylserine sulfhydrylase B or the second sulfhydrylase, O-acetylserine sulfhydrylase A (coded for by cysK), are cysteine prototrophs, but cysK cysM double mutants were found to require cysteine for growth. O-Acetylserine sulfhydrylase B was depressed by growth on a poor sulfur source, and depression was dependent upon both a functional cysB regulatory gene product and the internal inducer of the cysteine biosynthetic pathway, O-acetyl-L-serine. Furthermore, a cysBc strain, in which other cysteine biosynthetic enzymes cannot be fully repressed by growth on L-cystine, was found to be constitutive for O-acetylserine sulfhydrylase B as well. Thus O-acetylserine sulfhydrylase B is regulated by the same factors that control the expression of O-acetylserine sulfhydrylase A and other activities of the cysteine regulon. It is not clear why S. typhimurium has two enzymes whose physiological function appears to be to catalyze the same step of L-cysteine biosynthesis.
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PMID:Regulation of O-acetylserine sulfhydrylase B by L-cysteine in Salmonella typhimurium. 38 18

It has been determined from steady state kinetic studies using the sulfide ion selective electrode that O-acetylserine sulfhydrylase catalyzes a Bi Bi Ping Pong reaction between O-acetyl-L-serine and sulfide. Both O-acetyl-L-serine (OAS) and sulfide exhibit strong competitive substrate inhibition. A fit of all the data to the equation for the mechanism yields KOAS = 0.149 +/- 0.059 mM and KIOAS = 46.91 +/- 10.06 mM for O-acetyl-L-serine and KS2- = 0.066 +/- 0.004 mM and KIS2- = 0.013 +/- 0.006 mM for sulfide. Product inhibition studies varying either substrate at changing fixed levels of cysteine demonstrate that cysteine combines with enzyme at two places along the reaction sequence to produce inhibition with KiCys = 1.048 +/- 0.048 mM and KICys = 11.4 +/- 0.5 mM. Relatively high concentrations of acetate are required to produce inhibition and at least part of the acetate inhibition is due to ionic strength. However, the ability of acetate to reverse the spectral shift produced from the binding of O-acetyl-L-serine to enzyme and the isotope exchange between [14C]acetate and O-acetyl-L-serine does demonstrate that the O-acetyl-L-serine to acetate half-reaction is reversible. There is some doubt as to the specificity of acetate as a product inhibitor, since propionate can also be used to reverse the spectral shift. Spectral studies using ths spectral shift produced from binding O-acetyl-L-serine to enzyme confirms the assignment of a ping-pong mechanism since the spectral intermediate produced is alpha-aminoacrylic acid in Schiff base with pyridoxal phosphate and, therefore, the acetyl moiety has been beta eliminated. Isotope exchange has been demonstrated for both the O-acetyl-L-serine to acetate and sulfide to cysteine half-reactions which also confirms a ping-pong mechanism.
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PMID:A reaction mechanism from steady state kinetic studies for O-acetylserine sulfhydrylase from Salmonella typhimurium LT-2. 77 32

A 1,2,4-triazole resistant mutant of S. typhimurium has been isolated, in which serine transacetylase activity is seven times higher than in wild type. Partially purified serine transacetylase from a strain carrying the trz-312 mutation has kinetic properties which are virtually identical to those of the wild type enzyme and binds to O-acetylserine sulfhydrylase A to form a cysteine synthetase complex which is also indistinguishable from that found in wild type. Thus the increased activity of serine transacetylase associated with trz-312 appears to result from increased quantities of a kinetically normal, enzyme protein. Resistance to 1,2,4-triazole is probably due to the ability of trz-312 strains to synthesize O-acetyl-L-serine at a rapid enough rate to compensate for that utilized by the O-acetylserine triazolylase reaction. Genetic mapping experiments, using P1-mediated transduction, show that trz-312 is 91-99% linked to cysE, the structural gene for serine transacetylase. The results of three point crosses indicate that this mutation is located at one extreme end of the cysE locus, as would be expected for a promotor mutation.
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PMID:A mutation affecting expression of the gene coding for serine transacetylase in Salmonella typhimurium. 79 Jan 54

Saturation curves for O-acetylserine sulfhydrylase using the substrate analogues O-propionyl-L-serine, O-butyryl-L-serine, and beta-chloro-L-alanine all exhibit substrate inhibition and yield Km values comparable to O-acetyl-L-serine, except the O-butyryl derivative which has a Km 5-fold higher. Since all analogues are used as substrates and yield similar kinetic parameters in most cases, it is possible that they share a common intermediate. This evidence also suggests that specificity of O-acetylserine sulfhydrylase resides in the fact that the beta-substituted moiety on L-serine is a good leaving group. The overall rate equation for O-acetylserine sulfhydrylase was derived. A comparison of the numerical integration of the rate equation and an experimental time course is given.
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PMID:Overall mechanism and rate equation for O-acetylserine sulfhydrylase. 86 90

Forteen species (17 strains) of phototrophic bacteria as well as one strain of Thiobacillus denitrificans were tested for cysteine synthase and S-sulfocysteine synthase. All strains contain cysteine sythase active with O-acetylserine; only the Chromatiaceae, two species of the Rhodospirillaceae and T. denitrificans contain S-sulfocysteine synthase. In six species repression by different sulfur compounds in the medium was studied. In Chromatium vinosum, cysteine synthase was found to be constitutive, while in the Rhodospirillaceae tested the enzyme is repressed by sulfide. Thiosulfate had a derepressive effect in Rhodopseudomonas globiformis but strongly repressed cysteine synthase in R. sulfidophila and R. palustris. Cysteine had only moderate effects with the species tested.
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PMID:Cysteine and S-sulfocysteine biosynthesis in phototrophic bacteria. 96 65

The inhibition of Salmonella typhimurium by 1,2,4-triazole appears to be mediated through an effect on L-cysteine biosynthesis. O-Acetylserine sulfhydrylase A, the final enzyme in the L-cysteine biosynthetic pathway, was found to catalyze a reaction (triazolylase) between O-acetyl-L-serine and 1,2,4-triazole, giving 1,2,4-triazole-1-alanine as a product. In wild type S. typhimurium grown on 4 mM 1,2,4-triazole, 97% of the total O-acetyl-L-serine synthesized in vivo is incorporated into 1,2,4-triazole-1-alanine. 1,2,4-triazole also significantly lowers the levels of several of the enzymes necessary for sulfate reduction. This effect is presumably due to the ability of the inhibitor to lower intracellular concentrations of O-acetyl-L-serine, an inducer of these enzymes. Inhibition of growth is probably caused by L-cysteine starvation, arising from the decreased availability of the L-cysteine precursors, sulfide and O-acetyl-L-serine. Two 1,2,4-triazole-resistant strains bearing mutations in cysK, the structural gene for O-acetylserine sulfhydrylase A, incorporate only small quantities of O-acetyl-L-serine into 1,2,4-triazole-1-alanine in vivo. In vitro studies, using purified preparations of O-acetylserine sulfhydrylase A, revealed greater losses of triazolylase activity than sulfhydrylase activity in the enzymes from both cysK mutants. Resistance to 1,2,4-triazole apparently can arise from mutations leading to a preferential loss of triazolylase activity or from mutations which diminish both activities to the extent that high concentrations of O-acetyl-L-serine and sulfide accumulate behind the sulfhydrylase reaction.
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PMID:Studies on the mechanism of inhibition of Salmonella typhimurium by 1,2,4-triazole. 110 Jun 24

O-Acetylserine (thiol) lyase, the last enzyme in the cysteine biosynthetic pathway, was purified to homogeneity from spinach leaf chloroplasts. The enzyme has a molecular mass of 68,000 and consists of two identical subunits of Mr 35,000. The absorption spectrum obtained at pH 7.5 exhibited a peak at 407 nm due to pyridoxal phosphate, and addition of O-acetylserine induced a considerable modification of the spectrum. The pyridoxal phosphate content was found to be 1.1 per subunit of 35,000, and the chromophore was displaced from the enzyme by O-acetylserine, leading to a progressive inactivation of the holoenzyme. Upon gel filtration chromatography on Superdex 200, part of the chloroplastic O-acetylserine (thiol) lyase eluted in association with serine acetyltransferase at a position corresponding to a molecular mass of 310,000 (such a complex called cysteine synthase has been characterized in bacteria). The activity of O-acetylserine (thiol) lyase was optimum between pH 7.5 and 8.5. The apparent Km for O-acetylserine was 1.3 mM and for sulfide was 0.25 mM. The calculated activation energy was 12.6 kcal/mol at 10 mM O-acetylserine. The overall amino-acid composition of spinach chloroplast O-acetylserine (thiol) lyase was different than that determined for the same enzyme (cytosolic?) obtained from a crude extract of spinach leaves. A polyclonal antibody prepared against the chloroplastic O-acetylserine (thiol) lyase exhibited a very low cross-reactivity with a preparation of mitochondrial matrix and cytosolic proteins suggesting that the chloroplastic isoform was distinct from the mitochondrial and cytosolic counterparts.
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PMID:Purification and characterization of O-acetylserine (thiol) lyase from spinach chloroplasts. 137 15

Cysteine synthase (CSase) [O-acetyl-L-serine acetate-lyase (adding hydrogen sulfide), EC 4.2.99.8] catalyzes the formation of L-cysteine, the key step in sulfur assimilation in plants, from O-acetyl-L-serine and hydrogen sulfide. We report here the isolation and characterization of cDNA clones encoding cysteine synthase from spinach (Spinacia oleracea L.). Internal peptide sequences were obtained from V8 protease-digested fragments of purified CSase. A lambda gt10 cDNA library was constructed from poly(A)+ RNA of young green leaves of spinach. Screening with two synthetic mixed nucleotides encoding the partial peptide sequences revealed 19 positively hybridized clones among 2 x 10(5) clones. Nucleotide sequence analysis of two independent cDNA clones revealed a continuous open reading frame encoding a polypeptide of 325 amino acids with a calculated molecular mass of 34,185 Da. Sequence comparison of the deduced amino acids revealed 53% identity with CSases of Escherichia coli and Salmonella typhimurium. Sequence homology was also observed with other metabolic enzymes for amino acids in bacteria and yeast and with rat hemoprotein H-450. A bacterial expression vector was constructed and could genetically complement an E. coli auxotroph that lacks CSases. The accumulation of functionally active spinach CSase in E. coli was also demonstrated by immunoblotting and assaying enzymatic activity. Southern hybridization analysis showed the presence of two to three copies of the cDNA sequence in the genome of spinach. RNA blot hybridization suggested constitutive expression in leaves and roots of spinach.
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PMID:Molecular cloning and bacterial expression of cDNA encoding a plant cysteine synthase. 151 33

beta-(Isoxazolin-5-on-2-yl)alanine (BIA), a biosynthetic precursor of the neurotoxic amino acid beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (ODAP), was confirmed to be derived from O-acetyl-L-serine (OAS) and isoxazolin-5-one by cysteine synthase in higher plants. Some properties of this enzyme in the biosynthesis of BIA are described.
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PMID:Biosynthesis of beta-(isoxazolin-5-on-2-yl)alanine, the precursor of the neurotoxic amino acid beta-N-oxalyl-L-alpha,beta-diaminopropionic acid. 181 34

cDNA clones coding for hemoprotein H-450 were isolated from a rat liver cDNA library using anti-H-450 antibody. The molecular weight calculated from the deduced amino acid sequence comprising 547 amino acid residues was 60,085. The N-terminal sequence and a partial internal amino acid sequence of purified H-450, which were determined chemically, were both found in the amino acid sequence of H-450 deduced from the nucleotide sequence. H-450 mRNA is expressed in liver, kidney, and brain. A homology search of amino acid sequences indicated that H-450 shows no homology with cytochrome P-450, but shows significant homology with bacterial O-acetylserine (thiol)-lyases. However, H-450 has no O-acetylserine (thiol)-lyase activity.
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PMID:Molecular cloning and sequence analysis of cDNA coding for rat liver hemoprotein H-450. 208 36


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