Plasticity of the Tryptophan Synthase Active Site Probed by31P NMR Spectroscopy

Schnackerz, Klaus D.; Mozzarelli, Andrea

doi:10.1074/jbc.273.50.33247

Cited by 13 publications

(8 citation statements)

References 40 publications

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“…In contrast, the formation of the external aldimine of histidine at the b site did not trigger any conformational change, as evidenced by the absence of variation of the emission of aTrp129. These findings were later confirmed by X-ray crystal structures of several TS complexes (see below) and are in agreement with observed changes of 31 P NMR of the phosphate of the coenzyme, at different stages of the catalytic pathway [75]. The presence of distinct conformations of the b-subunit was also suggested by 15 N-heteronuclear single-quantum coherence NMR in the presence of 1-15 N-L-tryptophan [60].…”

Section: Spectroscopic Evidence Of Regulation-linked Conformational Tsupporting

confidence: 77%

Tryptophan synthase: a mine for enzymologists

Raboni

Bettati

Mozzarelli

2009

Cell. Mol. Life Sci.

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Tryptophan synthase is a pyridoxal 5'-phosphate-dependent alpha(2)beta(2) complex catalyzing the last two steps of tryptophan biosynthesis in bacteria, plants and fungi. Structural, dynamic and functional studies, carried out over more than 40 years, have unveiled that: (1) alpha- and beta-active sites are separated by about 20 A and communicate via the selective stabilization of distinct conformational states, triggered by the chemical nature of individual catalytic intermediates and by allosteric ligands; (2) indole, formed at alpha-active site, is intramolecularly channeled to the beta-active site; and (3) naturally occurring as well as genetically generated mutants have allowed to pinpoint functional and regulatory roles for several individual amino acids. These key features have made tryptophan synthase a text-book case for the understanding of the interplay between chemistry and conformational energy landscapes.

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Section: Spectroscopic Evidence Of Regulation-linked Conformational Tsupporting

confidence: 77%

Tryptophan synthase: a mine for enzymologists

Raboni

Bettati

Mozzarelli

2009

Cell. Mol. Life Sci.

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“…The addition of the substrate, serine, results in fluorescent changes that are consistent with the formation of an external aldimine and the reported absence of tritium washout from the ␣ carbon of serine in the absence of homocysteine. In the related enzymes tryptophan synthase (26) and O-acetylserine sulfhydrylase (22), the corresponding external aldimines rather than the elimination product, ␣-aminoacrylate, are also observed in the presence of serine alone. This is in contrast to the reported formation of the ␣-aminoacrylate product in heme-free cystathionine ␤-synthase in the crystalline state (11).…”

Section: Oxidation State Changes In the Heme Modulate Thementioning

confidence: 99%

Pyridoxal Phosphate Binding Sites Are Similar in Human Heme-dependent and Yeast Heme-independent Cystathionine β-Synthases

Kabil¹,

Toaka²,

LoBrutto

et al. 2001

Journal of Biological Chemistry

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Two classes of cystathionine ␤-synthases have been identified in eukaryotes, the heme-independent enzyme found in yeast and the heme-dependent form found in mammals. Both classes of enzymes catalyze a pyridoxal phosphate (PLP)-dependent condensation of serine and homocysteine to produce cystathionine. The role of the heme in the human enzyme and its location relative to the PLP in the active site are unknown.31 P NMR spectroscopy revealed that spin-lattice relaxation rates of the phosphorus nucleus in PLP are similar in both the paramagnetic ferric (T 1 ‫؍‬ 6.34 ؎ 0.01 s) and the diamagnetic ferrous (T 1 ‫؍‬ 5.04 ؎ 0.06 s) enzyme, suggesting that the two cofactors are not proximal to each other. This is also supported by pulsed EPR studies that do not provide any evidence for strong or weak coupling between the phosphorus nucleus and the ferric iron. However, the 31 P signal in the reduced enzyme moved from 5.4 to 2.2 ppm, and the line width decreased from 73 to 16 Hz, providing the first structural evidence for transmission to the active site of an oxidation state change in the heme pocket. These results are consistent with a regulatory role for the heme as suggested by previous biochemical studies from our laboratory. The 31 P chemical shifts of the resting forms of the yeast and human enzymes are similar, suggesting that despite the difference in their heme content, the microenvironment of the PLP is similar in the two enzymes. The addition of the substrate, serine, resulted in an upfield shift of the phosphorus resonance in both enzymes, signaling formation of reaction intermediates. The resting enzyme spectra were recovered following addition of excess homocysteine, indicating that both enzymes retained catalytic activity during the course of the NMR experiment.Cystathionine ␤-synthase catalyzes the condensation of serine and homocysteine to form cystathionine in a pyridoxal phosphate (PLP 1 )-dependent reaction. The mammalian enzyme is novel in its dependence on a second cofactor, heme, which distinguishes it from all other members of the PLP family of enzymes in which this combination of cofactors is not seen (1). In addition, the mammalian enzyme is allosterically activated by S-adenosylmethionine (2), a regulatory feature that is lacking in the related yeast enzyme (3). The reaction is postulated to involve a series of PLP-bound intermediates that are analogous to other PLP enzymes that catalyze ␤-replacement reactions (Fig. 1). Thus, the addition of serine results in a transaldimination reaction in which the Schiff base-forming lysine in the internal aldimine is replaced by serine to form the external aldimine. Binding of the second substrate, homocysteine, is followed by abstraction of the ␣-proton of serine and ␤-elimination of water to form the ␣-aminoacrylate intermediate, which is poised for nucleophilic addition by the thiolate of homocysteine. A second transaldimination reaction results in product release and regeneration of the resting enzyme. Fluorescence spectroscopy provides evidence for thi...

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“…This assumes that the substrate carboxylate group and active-site side-chain functional groups are ionized, as found spectroscopically for functional groups in other enzymes (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). The pyruvoyl dependent histidine decarboxylase does place its substrate carboxylate in a nonpolar environment (19), and nonpolar binding sites were successfully employed in the generation of PLP-dependent catalytic antibodies (20).…”

mentioning

confidence: 99%

Computational Studies on Nonenzymatic and Enzymatic Pyridoxal Phosphate Catalyzed Decarboxylations of 2-Aminoisobutyrate

Toney

2001

Biochemistry

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A computational study of nonenzymatic and enzymatic pyridoxal phosphate-catalyzed decarboxylation of 2-aminoisobutyrate (AIB) is presented. Four prototropic isomers of a model aldimine between AIB and 5′-deoxypyridoxal, with acetate interacting with the pyridine nitrogen, were employed in calculations of both gas phase and water model (PM3 and PM3-SM3) decarboxylation reaction paths. Calculations employing the transition state structures obtained for the four isomers allow the demonstration of stereoelectronic effects in transition state stabilization as well as a separation of the contributions of the Schiff base and pyridine ring moieties to this stabilization. The unprotonated Schiff base contribution (∼16 kcal/mol) is larger than that of the pyridine ring even when it is protonated (∼10 kcal/mol), providing an explanation of the catalytic power of pyruvoyl-dependent amino acid decarboxylases. An active site model of dialkylglycine decarboxylase was constructed and validated, and enzymatic decarboxylation reaction paths were calculated. The reaction coordinate is shown to be complex, with proton transfer from Lys272 to the coenzyme C4′ likely simultaneous with CR-CO 2 -bond cleavage. The proposed concerted decarboxylation/proton-transfer mechanism provides a simple explanation for the observed specificity of this enzyme toward oxidative decarboxylation.Pyridoxal phosphate (PLP) 1 dependent enzymes are ubiquitous and central to a wide variety of anabolic and catabolic pathways in the metabolism of nitrogen-containing compounds. The first, obligatory chemical step in all PLP dependent enzymatic reactions is formation of a Schiff base between the coenzyme aldehydic and substrate amino groups (Scheme 1). This common intermediate is termed the "external aldimine" in the literature. The general utility of PLP stems from its ability to stabilize carbanions formed adjacent to the Schiff base in the external aldimine intermediate through delocalization into the extended π bonded system (i.e., Schiff base and pyridine ring).Dunathan (32) proposed that the common fundamental mechanism that PLP dependent enzymes use to control reaction specificity is specific binding of the external aldimine intermediate such that the bond to be broken is oriented parallel to the p orbitals of the π-bonded system. This orientation maximizes orbital overlap between the nascent p orbital on CR and the π system in the transition state, thereby minimizing transition state energy. The magnitude of these proposed stereoelectronic effects have never been addressed.Dialkylglycine decarboxylase (DGD) is an intriguing PLP dependent enzyme that catalyzes two mechanistically different reactions: oxidative decarboxylation of 2,2-dialkylgycines in the first half-reaction, and transamination of R-keto acids in the second. Several DGD structures have been solved by X-ray crystallography (1-4).The active site structure shows that, to be bound productively for decarboxylation, a carboxylate group would make multiple favorable hydrogen bonding an...

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Plasticity of the Tryptophan Synthase Active Site Probed by31P NMR Spectroscopy

Cited by 13 publications

References 40 publications

Tryptophan synthase: a mine for enzymologists

Tryptophan synthase: a mine for enzymologists

Pyridoxal Phosphate Binding Sites Are Similar in Human Heme-dependent and Yeast Heme-independent Cystathionine β-Synthases

Computational Studies on Nonenzymatic and Enzymatic Pyridoxal Phosphate Catalyzed Decarboxylations of 2-Aminoisobutyrate

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