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

---

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

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.
...
PMID:A reaction mechanism from steady state kinetic studies for O-acetylserine sulfhydrylase from Salmonella typhimurium LT-2. 77 32

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.
...
PMID:Purification and characterization of O-acetylserine (thiol) lyase from spinach chloroplasts. 137 15

S-Sulfocysteine synthase was isolated from Salmonella typhimurium LT-2 to homogeneous form with polyacrylamide gel electrophoresis. The molecular weight of this enzyme was determined to be ca. 55,000. The enzyme consisted of two identically sized subunits, and it contained one pyridoxal phosphate per subunit. The enzyme catalyzed the biosynthesis of cysteine or S-methylcysteine from sulfide or methanethiol and O-acetylserine, respectively, in addition to the formation of S-sulfocysteine from thiosulfate and O-acetylserine. The enzyme is identical to cysteine synthase B. The intracellular level of this enzyme was regulated by lesser extents of the same factors as those effective for cysteine synthase A.
...
PMID:Enzymatic proof for the identity of the S-sulfocysteine synthase and cysteine synthase B of Salmonella typhimurium. 637 37

Comparison of seven deduced amino acid sequences of cysteine synthase (O-acetyl-L-serine (thiol)-lyase, EC 4.2.99.8) from plants and bacteria disclosed the presence of 12 conserved Lys residues, which can be candidates for a functional binding site for pyridoxal phosphate cofactor. These 12 conserved Lys residues in a cDNA clone encoding spinach cysteine synthase A were replaced with Gly by oligonucleotide-directed in vitro mutagenesis. These Lys-->Gly mutated cDNAs were transferred into Escherichia coli NK3, a cysteine auxotroph lacking both cysteine synthase loci, cysK and cysM. One mutant replaced at Lys-49 could not complement the cysteine requirement of NK3, whereas other mutants and wild-type clone could. No enzymatic activity of cysteine synthase A was detected either in the cell-free extracts of E. coli NK3 transformed with the Lys-49 mutant. These results indicated that Lys-49 is a functional residue for the catalytic activity of cysteine synthase. This Lys residue is conserved in other evolutionarily related amino acid-metabolizing enzymes catalyzing reactions involving the beta-carbon of amino acids.
...
PMID:Determination of a functional lysine residue of a plant cysteine synthase by site-directed mutagenesis, and the molecular evolutionary implications. 834 14

The A-isozyme of O-acetylserine sulfhydrylase, a pyridoxal phosphate-dependent enzyme isolated from Salmonella typhimurium catalyzes the synthesis of L-cysteine from O-acetyl-L-serine and sulfide. The pyridoxal form of the enzyme has been crystallized in two different forms. One form is in the orthorhombic space group P2(1)2(1)2(1) with cell constants a = 144.4 A, b = 96.9 A and c = 54.3 A and contains two monomers each of molecular weight 34,000 per asymmetric unit. The second form is in a hexagonal space group with unit cell dimensions a = b = 115 A, and c = 348 A and contains two 68,000 dimers per asymmetric unit. Complete native enzyme data sets have been collected for both crystal forms using an R-Axis II detector. A search for suitable heavy-atom derivatives is underway. Although both crystal forms diffract X-rays to better than 2.5 A, the orthorhombic form is more suited to a detailed structural analysis due to the extended lifetime in the X-ray beam and the relative size of the unit cell.
...
PMID:Crystallization and preliminary X-ray data for the A-isozyme of O-acetylserine sulfhydrylase from Salmonella typhimurium. 851 70

The cysteine synthase gene (cysK) from Flavobacterium K3-15 was cloned and sequenced. The gene exhibits 30-50% identity to known cysteine synthases on both the DNA and the amino acid levels. The pyridoxal phosphate binding site of the enzyme is part of a conserved motif comprising seven amino acids (SIKDRIA). The lys31 residue of the flavobacterial enzyme is conserved in all known cysteine synthases. The cysK gene from Flavobacterium K3-15 was heterologously expressed and the gene product identified by immunoblotting and determination of the enzyme activity.
...
PMID:Isolation of a gene encoding cysteine synthase from Flavobacterium K3-15. 886 84

Our studies of cystathionine beta-synthase from Saccharomyces cerevisiae (yeast) are aimed at clarifying the cofactor dependence and catalytic mechanism and obtaining a system for future investigations of the effects of mutations that cause human disease (homocystinuria or coronary heart disease). We report methods that yielded high expression of the yeast gene in Escherichia coli and of purified yeast cystathionine beta-synthase. The absorption and circular dichroism spectra of the homogeneous enzyme were characteristic of a pyridoxal phosphate enzyme and showed the absence of heme, which is found in human and rat cystathionine beta-synthase. The absence of heme in the yeast enzyme facilitates spectroscopic studies to probe the catalytic mechanism. The reaction of the enzyme with L-serine in the absence of L-homocysteine produced the aldimine of aminoacrylate, which absorbed at 460 nm and had a strong negative circular dichroism band at 460 nm. The formation of this intermediate from the product, L-cystathionine, demonstrates the partial reversibility of the reaction. Our results establish the overall catalytic mechanism of yeast cystathionine beta-synthase and provide a useful system for future studies of structure and function. The absence of heme in the functional yeast enzyme suggests that heme does not play an essential catalytic role in the rat and human enzymes. The results are consistent with the absence of heme in the closely related enzymes O-acetylserine sulfhydrylase, threonine deaminase, and tryptophan synthase.
...
PMID:Yeast cystathionine beta-synthase is a pyridoxal phosphate enzyme but, unlike the human enzyme, is not a heme protein. 1076 67

Our studies of the reaction mechanism of cystathionine beta-synthase from Saccharomyces cerevisiae (yeast) are facilitated by the spectroscopic properties of the pyridoxal phosphate coenzyme that forms a series of intermediates in the reaction of L-serine and L-homocysteine to form L-cystathionine. To characterize these reaction intermediates, we have carried out rapid-scanning stopped-flow and single-wavelength stopped-flow kinetic measurements under pre-steady-state conditions, as well as circular dichroism and fluorescence spectroscopy under steady-state conditions. We find that the gem-diamine and external aldimine of aminoacrylate are the primary intermediates in the forward half-reaction with L-serine and that the external aldimine of aminoacrylate or its complex with L-homocysteine is the primary intermediate in the reverse half-reaction with L-cystathionine. The second forward half-reaction of aminoacrylate with L-homocysteine is rapid. No primary kinetic isotope effect was obtained in the forward half-reaction with L-serine. The results provide evidence (1) that the formation of the external aldimine of L-serine is faster than the formation of the aminoacrylate intermediate, (2) that aminoacrylate is formed by the concerted removal of the alpha-proton and the hydroxyl group of L-serine, and (3) that the rate of the overall reaction is rate-limited by the conversion of aminoacrylate to L-cystathionine. We compare our results with cystathionine beta-synthase with those of related investigations of tryptophan synthase and O-acetylserine sulfhydrylase.
...
PMID:The reaction of yeast cystathionine beta-synthase is rate-limited by the conversion of aminoacrylate to cystathionine. 1153 64

The cysK gene encoding a cysteine synthase of Geobacillus stearothermophilus V was overexpressed in E. coli and the recombinant protein was purified and characterized. The enzyme is a thermostable homodimer (32 kDa/monomer) belonging to the beta family of pyridoxal phosphate (PLP)-dependent enzymes. UV-visible spectra showed absorption bands at 279 and 410 nm. The band at 279 nm is due to tyrosine residues as the enzyme lacks tryptophan. The 410 nm band represents absorption of the coenzyme bound as a Schiff base to a lysine residue of the protein. Fluorescence characteristics of CysK's Schiff base were influenced by temperature changes suggesting different local structures at the cofactor binding site. The emission of the Schiff base allowed the determination of binding constants for products at both 20 degrees C and 50 degrees C. At 50 degrees C and in the absence of sulphide the enzyme catalyzes the decomposition of O-acetyl-l-serine to pyruvate and ammonia. At 20 degrees C, however, a stable alpha-aminoacrylate intermediate is formed.
...
PMID:Biochemical characterization of a thermostable cysteine synthase from Geobacillus stearothermophilus V. 1530 37

Cysteine biosynthetic genes are up-regulated in the persistent phase of Mycobacterium tuberculosis, and the corresponding enzymes are therefore of interest as potential targets for novel antibacterial agents. cysK1 is one of these genes and has been annotated as coding for an O-acetylserine sulfhydrylase. Recombinant CysK1 is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the conversion of O-acetylserine to cysteine. The crystal structure of the enzyme was determined to 1.8A resolution. CysK1 belongs to the family of fold type II PLP enzymes and is similar in structure to other O-acetylserine sulfhydrylases. We were able to trap the alpha-aminoacrylate reaction intermediate and determine its structure by cryocrystallography. Formation of the aminoacrylate complex is accompanied by a domain rotation resulting in active site closure. The aminoacrylate moiety is bound in the active site via the covalent linkage to the PLP cofactor and by hydrogen bonds of its carboxyl group to several enzyme residues. The catalytic lysine residue is positioned such that it can protonate the Calpha-carbon atom of the aminoacrylate only from the si-face, resulting in the formation of L-cysteine. CysK1 is competitively inhibited by a four-residue peptide derived from the C-terminal of serine acetyl transferase. The crystallographic analysis reveals that the peptide binds to the enzyme active site, suggesting that CysK1 forms an bi-enzyme complex with serine acetyl transferase, in a similar manner to other bacterial and plant O-acetylserine sulfhydrylases. The structure of the enzyme-peptide complex provides a framework for the design of strong binding inhibitors.
...
PMID:Structural insights into catalysis and inhibition of O-acetylserine sulfhydrylase from Mycobacterium tuberculosis. Crystal structures of the enzyme alpha-aminoacrylate intermediate and an enzyme-inhibitor complex. 1756 78


1 2 Next >>

---