pH Dependence of the absorbance and phosphorus-31 NMR spectra of O-acetylserine sulfhydrylase in the absence and presence of O-acetyl-L-serine

Cook, Paul F.; Hara, Shinichi; Nalabolu, Srinivasa; Schnackerz, Klaus D.

doi:10.1021/bi00123a013

Cited by 64 publications

(121 citation statements)

References 17 publications

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“…Although titrations of the N77D, Q147A, and Q147E mutants with O-acetylserine yielded no detectable ␣-aminoacrylate intermediate, these mutants still catalyze cysteine formation with 1000-fold lower turnover rates compared with wild-type enzyme. In the OASS reaction, formation of the ␣-aminoacrylate intermediate is the rate-limiting step, and the intermediate decays with time in the absence of sulfur (20,46,47). The lack of detectable intermediate in the N77D, Q147A, and Q147E mutants indicates that the rate of intermediate formation is now slower than the dissociation rate, consistent with an increased K d value for O-acetylserine.…”

Section: Discussionmentioning

confidence: 71%

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Molecular Basis of Cysteine Biosynthesis in Plants

Bonner

Cahoon

Knapke

et al. 2005

Journal of Biological Chemistry

171

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Section: Discussionmentioning

confidence: 71%

“…1C, step 4). The active site lysine reacts with this intermediate, releasing cysteine and regenerating the Schiff base (20,21). Site-directed mutagenesis of Salmonella typhimurium OASS (StOASS) confirmed the importance of the lysine (22) and the contribution of an active site serine to stabilizing the pyridoxal ring in the reaction (23).…”

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confidence: 90%

Molecular Basis of Cysteine Biosynthesis in Plants

Bonner

Cahoon

Knapke

et al. 2005

Journal of Biological Chemistry

171

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“…2A). The 460 nm band is likely due to the aldimine of aminoacrylate (E-AA in Scheme 1), which has been detected in the reaction of O-acetylserine sulfhydrylase with O-acetyl-L-serine (32)(33)(34) and of D-serine dehydratase with D-serine (35). The 330 nm shoulder may be due to a different tautomer of E-AA, which is the predominant intermediate in the reaction of the closely related tryptophan synthase with L-serine (36).…”

Section: Resultsmentioning

confidence: 99%

Yeast Cystathionine β-Synthase Is a Pyridoxal Phosphate Enzyme but, Unlike the Human Enzyme, Is Not a Heme Protein

Jhee

McPhie

Miles

2000

Journal of Biological Chemistry

137

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Our studies of cystathionine ␤-synthase from Saccharomyces cerevisiae (yeast) are aimed at (1) clarifying the cofactor dependence and catalytic mechanism and (2) 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 ␤-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 ␤-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 ␤-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.Elevated plasma homocysteine is an important risk factor in coronary heart disease and other human diseases (1-3). One of the two major routes for detoxication of homocysteine is the pyridoxal phosphate (PLP) 1 -dependent ␤-replacement reactionThe deduced sequences of human (4,5), rat (6), and Saccharomyces cerevisiae (yeast) (7,8) CBS are similar. The finding that human CBS complements the cysteine auxotrophy of a yeast strain lacking CBS (5) demonstrates the functional conservation of the human and yeast genes.The remarkable observation that the sequence of rat CBS (6) is identical to the sequence of rat hemoprotein H-450 (9) led to the discovery that rat and human CBS contain both PLP and heme (10). Heme may play a role in redox regulation of the human enzyme and in binding homocysteine (11,12). Although yeast CBS has been purified to homogeneity (13), the absorption spectrum and cofactor content have not been reported. Here, we demonstrate that purified yeast CBS contains PLP but not heme. Because the absence of heme facilitates spectroscopic studies of the PLP and of enzyme-substrate intermediates, we are able to demonstrate directly that CBS converts L-serine to an aminoacrylate intermediate, as expected for a PLP enzyme that catalyzes a ␤-replacement reaction (14,15). EXPERIMENTAL PROCEDURESChemicals-L-Cystathionine and L-serine were from Fluka. ␦-Aminolevulinic acid, L-homocysteine thiolactone, aprotinin, pepstatin A, leupeptin, benzamidine hydrochloride, TPCK, TLCK, and PMSF were from S...

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“…4b). This band is attributed to the ketoenamine tautomer of the ␣-aminoacrylate absorbing at 450 -470 nm, as observed in O-acetylserine sulfhydrylase (22) and cystathionine ␤-synthase (23,24). This result indicates a negligible influence of IAG and GP on the equilibrium distribution of ␤-intermediates.…”

Section: Resultsmentioning

confidence: 69%

Allosteric Communication of Tryptophan Synthase

Biase

Tramonti

Bettati

et al. 2001

Journal of Biological Chemistry

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The ␣ 2 ␤ 2 tryptophan synthase complex is a model enzyme for understanding allosteric regulation. We report the functional and regulatory properties of the ␤S178P mutant. Ser-178 is located at the end of helix 6 of the ␤ subunit, belonging to the domain involved in intersubunit signaling. The carbonyl group of ␤Ser-178 is hydrogen bonded to Gly-181 of loop 6 of the ␣ subunit only when ␣ subunit ligands are bound. An analysis by molecular modeling of the structural effects caused by the ␤S178P mutation suggests that the hydrogen bond involving ␣Gly-181 is disrupted as a result of localized structural perturbations. The ratio of ␣ to ␤ subunit concentrations was calculated to be 0.7, as for the wild type, indicating the maintenance of a tight ␣؊␤ complex. Both the activity of the ␣ subunit and the inhibitory effect of the ␣ subunit ligands indole-3-acetylglycine and D,L-␣-glycerol-3-phosphate were found to be the same for the mutant and wild type enzyme, whereas the ␤ subunit activity of the mutant exhibited a 2-fold decrease. In striking contrast to that observed for the wild type, the allosteric effectors indole-3-acetylglycine and D,L-␣-glycerol-3-phosphate do not affect the ␤ activity. Accordingly, the distribution of L-serine intermediates at the ␤-site, dominated by the ␣-aminoacrylate, is only slightly influenced by ␣ subunit ligands. Binding of sodium ions is weaker in the mutant than in the wild type and leads to a limited increase of the amount of the external aldimine intermediate, even at high pH, whereas binding of cesium ions exhibits the same affinity and effects as in the wild type, leading to an increase of the ␣-aminoacrylate tautomer absorbing at 450 nm. Crystals of the ␤S178P mutant were grown, and their functional and regulatory properties were investigated by polarized absorption microspectrophotometry. These findings indicate that (i) the reciprocal activation of the ␣ and ␤ activity in the ␣2␤2 complex with respect to the isolated subunits results from interactions that involve residues different from ␤Ser-178 and (ii) ␤Ser-178 is a critical residue in ligand-triggered signals between ␣ and ␤ active sites.Allosteric proteins are key elements of cellular regulation, because they allow for a fine adaptation of the living organisms to changes in metabolite concentration and physico-chemical conditions. Tryptophan synthase is an allosteric enzyme, composed of two ␣ and two ␤ subunits, catalyzing the last two steps in the biosynthesis of L-tryptophan (1-3) (Fig. 1). The ␣ active site cleaves indole-3-glycerol phosphate into indole and D-glyceraldehyde-3-phosphate. The ␤ active site contains a pyridoxal 5Ј-phosphate molecule bound to Lys-87 via a Schiff base and catalyzes the replacement of the ␤-hydroxyl of L-serine with indole. The latter compound is intramolecularly channeled from the ␣ site via a hydrophobic tunnel connecting the ␣ and ␤ active sites, as proposed on the basis of the first crystal structure of the enzyme (4). The ␣ and ␤ activities are reciprocally modulated. Extensive functi...

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pH Dependence of the absorbance and phosphorus-31 NMR spectra of O-acetylserine sulfhydrylase in the absence and presence of O-acetyl-L-serine

Cited by 64 publications

References 17 publications

Molecular Basis of Cysteine Biosynthesis in Plants

Molecular Basis of Cysteine Biosynthesis in Plants

Yeast Cystathionine β-Synthase Is a Pyridoxal Phosphate Enzyme but, Unlike the Human Enzyme, Is Not a Heme Protein

Allosteric Communication of Tryptophan Synthase

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