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)

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

The pH dependence of kinetic parameters using natural and alternative reactants was determined in order to obtain information on the chemical mechanisms of the A and B isozymes of O-acetylserine sulfhydrylase (OASS) from Salmonella typhimurium. A general mechanism is proposed for OASS in which OAS binds with its alpha-amine unprotonated to carry out a nucleophilic attack on C4' of the protonated Schiff base and with the acetyl carbonyl hydrogen-bonded to a protonated enzyme group (or a water molecule), which aids in the beta-elimination of acetate. The enzyme lysine that was in Schiff base linkage with the active site pyridoxal 5'-phosphate deprotonates the alpha-carbon in the beta-elimination reaction, and a proton is likely released with the acetate product. Sulfide likely binds as HS- to undergo nucleophilic attack on the alpha-aminoacrylate intermediate, followed by protonation of the alpha-carbon by the enzyme lysine. In OASS-A, HS- is hydrogen-bonded to the enzyme group that assists in the beta-elimination of acetate, but this is not the case for OASS-B. The pH independent equilibrium constant for the first half-reaction of OASS-A is 1.6 x 10(-3), while the second half-reaction is practically irreversible.
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PMID:Acid-base chemical mechanism of O-acetylserine sulfhydrylases-A and -B from pH studies. 754 74

The intrinsic fluorescence of the pyridoxal 5'-phosphate (PLP) enzyme O-acetylserine sulfhydrylase-A (OASS-A) was studied in order to gain insight into the structural basis for binding of substrates and products and for catalysis. Excitation of OASS-A with 298-nm light gives an emission spectrum with two maxima, 337 and 498 nm. OASS-A has two tryptophan residues, and the 337-nm maximum indicates that at least one of these is exposed somewhat to aqueous solvent. The 498-nm emission observed is due to fluorescence of the PLP Schiff base. Some of this long-wavelength fluorescence is likely due to direct excitation by incident radiation. However, the concomitant quenching of 340-nm emission and the enhancement of 498-nm emission observed upon reconstitution of apoenzyme with PLP support the conclusion that some of the long-wavelength emission is due to singlet-singlet transfer from at least one tryptophan residue to the PLP Schiff base. Enhancement of 498-nm fluorescence by either of the products, acetate or cysteine, of the enzymatic reaction without a quenching of 337-nm fluorescence is consistent with triplet-singlet transfer from one or both of the tryptophan residues to the PLP Schiff base. This would require a rigid environment for the tryptophan donor when the product is bound. However, a conformational change which affected principally the environment of the PLP Schiff base, resulting in a longer lifetime of its excited singlet state, would also increase the intensity of the 498-nm emission. Enhancement of OASS-A long-wavelength fluorescence by each product requires the unprotonated form of a different group on enzyme. Enhancement by acetate binding requires the unprotonated form of an enzyme group with a pK of 7 and is insensitive to substitution on the methyl group. L-Cysteine binding enhances 498-nm fluorescence when a group with a pK of 8 is unprotonated, and substitution at the thiol or the methylene bridge does not affect the enhancement elicited. Binding of L-cysteine to free enzyme (E) likely results in the formation of the external Schiff base accompanied by a conformational change giving fluorescence enhancement. The carboxylate moiety of acetate likely binds to the alpha-carboxylate subsite for amino acid reactants such as L-cysteine, resulting in a conformational change in the internal Schiff base and giving rise to the observed fluorescence enhancement. Data are interpreted in terms of the mechanism of OASS-A.
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PMID:Product binding to the alpha-carboxyl subsite results in a conformational change at the active site of O-acetylserine sulfhydrylase-A: evidence from fluorescence spectroscopy. 811 Jul 69

The resonance-stabilized quinonoid 5-mercapto-2-nitrobenzoate (TNB) is a substrate for O-acetylserine sulfhydrylase-A (OASS-A) and -B (OASS-B), giving rise to the product S-(3-carboxy-4-nitrophenyl)-L-cysteine (S-CNP-cysteine) as confirmed by ultraviolet-visible and 1H NMR spectroscopies. A comparison of the kinetics of OASS-A and OASS-B indicates that the mechanism proceeds predominantly via a bi-bi ping pong kinetic mechanism as suggested by an initial velocity pattern consisting of parallel lines at low concentrations of reactants, but competitive inhibition by both substrates as the reactant concentrations are increased. Thus, in the first half-reaction, O-acetyl-L-serine (OAS) or beta-chloro-L-alanine (BCA) is converted to alpha-aminoacrylate in Schiff base with the active site pyridoxal 5'-phosphate, while in the second half-reaction cysteine (with sulfide as the reactant) or S-CNP-cysteine (with TNB as the reactant) is formed. The ping pong mechanism is corroborated by a qualitative and quantitative analysis of product and dead-end inhibition. Product inhibition by acetate is S-parabolic noncompetitive. These data are consistent with acetate reversing the first half-reaction and producing more free enzyme to which acetate may also bind. Thus, there may be some randomness to the mechanism at high concentrations of the nucleophilic substrate.
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PMID:Kinetic mechanisms of the A and B isozymes of O-acetylserine sulfhydrylase from Salmonella typhimurium LT-2 using the natural and alternative reactants. 851 86

Static and time-resolved fluorescence of the internal aldimine of the pyridoxal 5'-phosphate (PLP)-dependent enzyme O-acetylserine sulfhydrylase (OASS) and those of free PLP, and the PLP-L-valine Schiff base have been measured to gain insight into the photophysics of PLP bound to OASS. Exciting at 330 nm, free coenzyme exhibits a band at 415 nm, whereas PLP-valine and OASS (also when excited at their absorbance maxima) exhibit a structured emission with a peak at 420 nm and shoulders at 490 and 530 nm. The emission bands at 420 and 490 nm are attributed to the enolimine and ketoenamine tautomers of the internal aldimine, respectively, while the 530 nm emission might arise from a dipolar species formed upon proton dissociation in the excited state. Time-resolved fluorescence of OASS (PLP-valine), excited at 412 nm (415 nm) and collected at lamda > 470 nm, indicates the presence of two components characterized by lifetimes (tau) of 0.6 (0.08) and 3.8 (1.55) ns with equal fractional intensity (f). In the presence of acetate the slow component dominates OASS emission with f of 0.98. Excitation at 350 nm as a function of emission wavelengths (400-560 nm) shows at least three components. The f of the slow component increases from 400 to 440 nm, then decreases, whereas the f of the intermediate and fast components behave in the opposite way. Results indicate that: (i) the fast component is associated with the emission at 530 nm; (ii) the slow component is associated with the emission at 420 nm; (iii) a fast additive component, characterized by a very short lifetime, is present on the blue side of the emission spectrum; (iv) the intermediate component results from overlapping contributions, including the emission of the band at 490 nm, that could not be resolved; (v) the increased emission at 490 nm, caused by acetate binding, is likely due to the stabilization of the ketoenamine tautomer induced by an increase in polarity of the active site microenvironment and/or a decrease in proton dissociation in the excited state; (vi) excitation at 330 nm, where the enolimine tautomer absorbs, leads to emission decays typical of the ketoenamine.
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PMID:Time-resolved fluorescence of O-acetylserine sulfhydrylase. 998 17

Covalent binding of L-methionine as an external aldimine to the pyridoxal 5'-phosphate-cofactor in the K41A mutant of O-acetylserine sulfhydrylase from Salmonella typhimurium induces a large conformational change in the protein. Methionine mimics the action of the substrate O-acetyl-L-serine during catalysis. The alpha-carboxylate moiety of L-methionine in external aldimine linkage with the active site pyridoxal 5'-phosphate forms a hydrogen bonding network to the "asparagine-loop" P67-T68-N69-G70 which adopts a different conformation than in the native protein. The side-chain nitrogen of Asn69 moves more than 7 A to make a hydrogen bond to the alpha-carboxylate group of the inhibitor. As the external aldimine is formed, the PLP tilts by 13 degrees along its longitudinal axis such that C4' moves toward the entrance to the active site and the side-chain of the methionine is directed toward the active site entrance. The local rearrangement acts as a trigger to induce a large global conformational change in the protein. A subdomain comprised of beta-strand 4, alpha-helix 3, beta-strand 5 and alpha-helix 4 moves towards the active site by a rotation of 7 degrees. This subdomain movement results in a reduction of the severe twist of its central beta-sheet and reduces the active site entrance to a small hole, giving access only to small molecules like sulfide, the second substrate, or acetate, the first product.
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PMID:Ligand binding induces a large conformational change in O-acetylserine sulfhydrylase from Salmonella typhimurium. 1045 98

Cysteine is the major source of fixed sulfur for the synthesis of sulfur-containing compounds in organisms of the Bacteria and Eucarya domains. Though pathways for cysteine biosynthesis have been established for both of these domains, it is unknown how the Archaea fix sulfur or synthesize cysteine. None of the four archaeal genomes sequenced to date contain open reading frames with identities to either O-acetyl-L-serine sulfhydrylase (OASS) or homocysteine synthase, the only sulfur-fixing enzymes known in nature. We report the purification and characterization of OASS from acetate-grown Methanosarcina thermophila, a moderately thermophilic methanoarchaeon. The purified OASS contained pyridoxal 5'-phosphate and catalyzed the formation of L-cysteine and acetate from O-acetyl-L-serine and sulfide. The N-terminal amino acid sequence has high sequence similarity with other known OASS enzymes from the Eucarya and Bacteria domains. The purified OASS had a specific activity of 129 micromol of cysteine/min/mg, with a K(m) of 500 +/- 80 microM for sulfide, and exhibited positive cooperativity and substrate inhibition with O-acetyl-L-serine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band at 36 kDa, and native gel filtration chromatography indicated a molecular mass of 93 kDa, suggesting that the purified OASS is either a homodimer or a homotrimer. The optimum temperature for activity was between 40 and 60 degrees C, consistent with the optimum growth temperature for M. thermophila. The results of this study provide the first evidence for a sulfur-fixing enzyme in the Archaea domain. The results also provide the first biochemical evidence for an enzyme with the potential for involvement in cysteine biosynthesis in the Archaea.
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PMID:O-Acetylserine sulfhydrylase from Methanosarcina thermophila. 1061 61

The O-acetylserine sulfhydrylase (OASS) from Salmonella typhimurium catalyzes a beta-replacement reaction in which the beta-acetoxy group of O-acetyl-L-serine (OAS) is replaced by bisulfide to give L-cysteine and acetate. The kinetic mechanism of OASS is ping-pong with a stable alpha-aminoacrylate intermediate. The enzyme is a homodimer with one pyridoxal 5'-phosphate (PLP) bound per subunit deep within the protein in a cleft between the N- and C-terminal domains of each of the monomers. All of the active site residues are contributed by a single subunit. The enzyme cycles through open and closed conformations as it catalyzes its reaction with structural changes largely limited to a subdomain of the N-terminal domain. The elimination of acetic acid from OAS is thought to proceed via an anti-E2 mechanism, and the only catalytic group identified to date is lysine 41, which originally participates in Schiff base linkage to PLP. The transition state for the elimination of acetic acid is thought to be asynchronous and earlier for Cbeta-O bond cleavage than for Calpha-H bond cleavage.
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PMID:Structure and mechanism of O-acetylserine sulfhydrylase. 1507 90

The gram-positive, thermophilic, acetogenic bacterium Moorella thermoacetica can reduce CO2 to acetate via the Wood-Ljungdahl (acetyl coenzyme A synthesis) pathway. This report demonstrates that, despite its classification as a strict anaerobe, M. thermoacetica contains a membrane-bound cytochrome bd oxidase that can catalyze reduction of low levels of dioxygen. Whole-cell suspensions of M. thermoacetica had significant endogenous O2 uptake activity, and this activity was increased in the presence of methanol or CO, which are substrates in the Wood-Ljungdahl pathway. Cyanide and azide strongly (approximately 70%) inhibited both the endogenous and CO/methanol-dependent O2 uptake. UV-visible light absorption and electron paramagnetic resonance spectra of n-dodecyl-beta-maltoside extracts of M. thermoacetica membranes showed the presence of a cytochrome bd oxidase complex containing cytochrome b561, cytochrome b595, and cytochrome d (chlorin). Subunits I and II of the bd oxidase were identified by N-terminal amino acid sequencing. The M. thermoacetica cytochrome bd oxidase exhibited cyanide-sensitive quinol oxidase activity. The M. thermoacetica cytochrome bd (cyd) operon consists of four genes, encoding subunits I and II along with two ABC-type transporter proteins, homologs of which in other bacteria are required for assembly of the bd complex. The level of this cyd operon transcript was significantly increased when M. thermoacetica was grown in the absence of added reducing agent (cysteine + H2S). Expression of a 35-kDa cytosolic protein, identified as a cysteine synthase (CysK), was also induced by the nonreducing growth conditions. The combined evidence indicates that cytochrome bd oxidase and cysteine synthase protect against oxidative stress and contribute to the limited dioxygen tolerance of M. thermoacetica.
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PMID:Cytochrome bd oxidase, oxidative stress, and dioxygen tolerance of the strictly anaerobic bacterium Moorella thermoacetica. 1574 50

O-Acetylserine sulfhydrylase is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the final step in the cysteine biosynthetic pathway in enteric bacteria and plants, the replacement of the beta-acetoxy group of O-acetyl-l-serine by a thiol to give l-cysteine. Two isozymes are found in Salmonella typhimurium, with the A-isozyme expressed under aerobic and the B-isozyme expressed under anaerobic conditions. The structure of O-acetylserine sulfhydrylase B has been solved to 2.3 A and exhibits overall a fold very similar to that of the A-isozyme. The main difference between the two isozymes is the more hydrophilic active site of the B-isozyme with two ionizable residues, C280 and D281, replacing the neutral residues S300 and P299, respectively, in the A-isozyme. D281 is above the re face of the cofactor and is within hydrogen-bonding distance to Y286, while C280 is located about 3.4 A from the pyridine nitrogen (N1) of the internal Schiff base. The B-isozyme has a turnover number (V/Et) 12.5-fold higher than the A-isozyme and an approximately 10-fold lower Km for O-acetyl-l-serine. Studies of the first half-reaction by rapid-scanning stopped-flow indicate a first-order conversion of the internal Schiff base to the alpha-aminoacrylate intermediate at any concentration of O-acetyl-l-serine. The Kd values for formation of the external Schiff base with cysteine and serine, obtained by spectral titration, are pH dependent and exhibit a pKa of 7.0-7.5 (for a group that must be unprotonated for optimum binding) with values, above pH 8.0, of about 3.0 and 30.0 mM, respectively. In both cases the neutral enolimine is favored at high pH. Failure to observe the pKa for the alpha-amines of cysteine and serine in the pKESB vs pH profile suggests a compensatory effect resulting from titration of a group on the enzyme with a pKa in the vicinity of the alpha-amine's pKa. The pH dependence of the first-order rate constant for decay of the alpha-aminoacrylate intermediate to give pyruvate and ammonia gives a pKa of about 9 for the active site lysine (K41), a pH unit higher than that of the A-isozyme. The difference in pH dependence of the pKESB for cysteine and serine, the higher pKa for K41, and the preference for the neutral species at high pH compared to the A-isozyme can be explained by titration of C280 to give the thiolate. Subtle conformational differences between O-acetylserine sulfhydrylase A and O-acetylserine sulfhydrylase B are detected by comparing the absorption and emission spectra of the internal aldimine in the absence and presence of the product acetate and of the external aldimine with l-serine. The two isozymes show a different equilibrium distribution of the enolimine and ketoenamine tautomers, likely as a result of a more polar active site for O-acetylserine sulfhydrylase B. The distribution of cofactor tautomers is dramatically affected by the ligation state of the enzyme. In the presence of acetate, which occupies the alpha-carboxylate subsite, the equilibrium between tautomers is shifted toward the ketoenamine tautomer, as a result of a conformational change affecting the structure of the active site. This finding, in agreement with structural data, suggests for the O-acetylserine sulfhydrylase B-isozyme a higher degree of conformational flexibility linked to catalysis.
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PMID:Structure, mechanism, and conformational dynamics of O-acetylserine sulfhydrylase from Salmonella typhimurium: comparison of A and B isozymes. 1758 14


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