Detection and identification of intermediates in the reaction of L-serine with Escherichia coli tryptophan synthase via rapid-scanning ultraviolet-visible spectroscopy

Drewe, William Frederick; Dunn, Michael F.

doi:10.1021/bi00336a027

Cited by 126 publications

(241 citation statements)

References 32 publications

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“…This is consistent with the lack of a primary kinetic isotope effect on aminoacrylate formation in yCBS (25). In contrast, a primary kinetic isotope effect on formation of an aminoacrylate intermediate is seen in both O-acetylserine sulfhydrylase and tryptophan synthase, which are fold II PLP enzymes that catalyze ␤-replacement reactions (20,29,30).…”

Section: Discussionsupporting

confidence: 78%

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Detection of Reaction Intermediates during Human Cystathionine β-Synthase-monitored Turnover and H2S Production

Yadav

Banerjee

2012

Journal of Biological Chemistry

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Section: Discussionsupporting

confidence: 78%

“…O-acetylserine sulfhydrylase (18), threonine deaminase (19), and tryptophan synthase (20). The PLP is tethered in the active site of human CBS via a Schiff base linkage with Lys-119 (Fig.…”

mentioning

confidence: 99%

Detection of Reaction Intermediates during Human Cystathionine β-Synthase-monitored Turnover and H2S Production

Yadav

Banerjee

2012

Journal of Biological Chemistry

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“…1A), indicating a rather high mobility of the phosphate around the C-O bond of the cofactor. This value is much smaller than that found for the internal aldimine of the E. coli enzyme in the presence of 200 mM Na ϩ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). These data suggest that the coenzyme experiences in the two enzyme forms different environment and/or a different rate of interconversion among an ensemble of conformations.…”

Section: P Nmr Of Pyridoxal 5ј-phosphate Of Tryptophan Synthase 33249mentioning

confidence: 63%

Plasticity of the Tryptophan Synthase Active Site Probed by31P NMR Spectroscopy

Schnackerz¹,

Mozzarelli²

1998

Journal of Biological Chemistry

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The functional properties of tryptophan synthase ␣ 2 ␤ 2 complex are modulated by a variety of allosteric effectors, including pH, monovalent cations, and ␣-subunit ligands. The dynamic properties of the ␤-active site were probed by 31 P NMR spectroscopy of the enzymebound coenzyme pyridoxal 5-phosphate. The 31 P NMR signal of the cofactor phosphate of the internal aldimine exhibits a single peak at 3.73 ppm with a line width of 12 Hz. In the presence of saturating concentrations of sodium ions, the 31 P signal shifts to 3.97 ppm concomitant with a change in line width to 35 Hz. The latter indicates that sodium ions decrease the conformational flexibility of the coenzyme. In the absence of ions, lowering pH leads to the appearance of a second peak at 4.11 ppm, the intensity of which decreases in the presence of cesium ions. Addition of L-serine in the presence of sodium ions leads to the formation of the external aldimine, the first metastable catalytic intermediate. The 31 P signal does not change its position, but a change in line width from 35 to 5 Hz is observed, revealing that this species is characterized by a considerable degree of rotational freedom around the coenzyme C-O bond. In the presence of L-serine and either cesium ions or the allosteric effector indole-3-acetylglycine, the accumulation of the second catalytic intermediate, ␣-aminoacrylate, is observed. The 31 P signal is centered at 3.73 ppm with a line width of 5 Hz, indicating that the phosphate group of the coenzyme in the external aldimine and the ␣-aminoacrylate exhibits the same flexibility but a slightly different state of ionization. Because the ␣-aminoacrylate intermediate but not the external aldimine triggers the allosteric signal to the ␣-subunit, other portions of the ␤-active site modify their dynamic properties in response to the progress of the catalytic process. A narrow line width was also observed for the quinonoid species formed by nucleophilic attack of indoline to the ␣-aminoacrylate. The 31 P signal moves downfield to 4.2 ppm, indicating a possible change of the ionization state of the phosphate group. Thus, the modification of either the ionization state of the coenzyme phosphate or its flexibility or both are, at least in part, responsible for the conformational events that accompany the catalytic process.

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“…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). Our results ( Fig.…”

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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Detection and identification of intermediates in the reaction of L-serine with Escherichia coli tryptophan synthase via rapid-scanning ultraviolet-visible spectroscopy

Cited by 126 publications

References 32 publications

Detection of Reaction Intermediates during Human Cystathionine β-Synthase-monitored Turnover and H2S Production

Detection of Reaction Intermediates during Human Cystathionine β-Synthase-monitored Turnover and H2S Production

Plasticity of the Tryptophan Synthase Active Site Probed by31P NMR Spectroscopy

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

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