pH Dependence of Tryptophan Synthase Catalytic Mechanism

Schiaretti, Francesca; Bettati, Stefano; Viappiani, Cristiano; Mozzarelli, Andrea

doi:10.1074/jbc.m401895200

Cited by 10 publications

(19 citation statements)

References 62 publications

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“…Two ionizable residues with pK a1 * 6 and pK a2 * 9 control the formation of a-aminoacrylate in the absence of monovalent cations or in the presence of sodium and potassium ions. In the presence of cesium ions, a single ionizable residue (pK a * 9) is involved in the formation of a-aminoacrylate [54]. This behavior is mainly attributable to effects on the fast phase of the external aldimine decay, since the rate of the slow process is essentially unaffected by pH and monovalent cations.…”

Section: B-reaction Kinetics and Identification Of Catalytic Residuesmentioning

confidence: 96%

“…Furthermore, the rate of the external aldimine decay is dependent on monovalent cations, following the order: [44,54]. The decay rate of the external aldimine decreases as pH increases [54]. Two ionizable residues with pK a1 * 6 and pK a2 * 9 control the formation of a-aminoacrylate in the absence of monovalent cations or in the presence of sodium and potassium ions.…”

Section: B-reaction Kinetics and Identification Of Catalytic Residuesmentioning

confidence: 98%

Section: B-reaction Kinetics and Identification Of Catalytic Residuesmentioning

confidence: 98%

“…The decay process to give the a-aminoacrylate species consists of a dominating relaxation followed by a slower phase, with small amplitude. A third phase of smaller amplitude is generally considered catalytically irrelevant and neglected [52][53][54][55]. The biphasic kinetics of the disappearance of the external aldimine suggests that the enzyme possesses two routes for formation of the a-aminoacrylate intermediate.…”

Section: B-reaction Kinetics and Identification Of Catalytic Residuesmentioning

confidence: 99%

“…The kinetics of stage I of the b-reaction (i.e., the conversion of the internal aldimine to the external aldimine and to a-aminoacrylate) has been the subject of extensive investigation via equilibrium, rapid mixing stopped-flow, and pressure and temperature jump relaxation studies [15,43,44,[49][50][51][52][53][54]. Upon binding of the b-substrate and in the absence of indole, the external aldimine transiently accumulates and then decays to an equilibrium mixture of external aldimine and a-aminoacrylate.…”

Section: B-reaction Kinetics and Identification Of Catalytic Residuesmentioning

confidence: 99%

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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: B-reaction Kinetics and Identification Of Catalytic Residuesmentioning

confidence: 96%

Section: B-reaction Kinetics and Identification Of Catalytic Residuesmentioning

confidence: 98%

Section: B-reaction Kinetics and Identification Of Catalytic Residuesmentioning

confidence: 98%

Section: B-reaction Kinetics and Identification Of Catalytic Residuesmentioning

confidence: 99%

Section: B-reaction Kinetics and Identification Of Catalytic Residuesmentioning

confidence: 99%

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Tryptophan synthase: a mine for enzymologists

Raboni

Bettati

Mozzarelli

2009

Cell. Mol. Life Sci.

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Exploring the pyridoxal 5′‐phosphate‐dependent enzymes

Mozzarelli

Bettati

2006

The Chemical Record

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Pyridoxal 5'-phosphate (PLP)-dependent enzymes represent about 4% of the enzymes classified by the Enzyme Commission. The versatility of PLP in carrying out a large variety of reactions exploiting the electron sink effect of the pyridine ring, the conformational changes accompanying the chemical steps and stabilizing distinct catalytic intermediates, and the spectral properties of the different coenzyme-substrate derivatives signaling the reaction progress, are some of the features that have attracted our interest to investigate the structure-dynamics-function relationships of PLP-dependent enzymes. To this goal, an integrated approach combining biochemical, biophysical, computational, and molecular biology methods was used. The extensive work carried out on two enzymes, tryptophan synthase and O-acetylserine sulfhydrylase, is presented and discussed as representative of other PLP-dependent enzymes we have investigated. Finally, perspectives of PLP-dependent enzymes functional genomics and drug targeting highlight the continuous novelty of an "old" class of enzymes.

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Regulation of human serine racemase activity and dynamics by halides, ATP and malonate

et al. 2014

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D-Serine is a non-proteinogenic amino acid that acts as a co-agonist of the NMDA receptors in the central nervous system. D-Serine is produced by human serine racemase (hSR), a homodimeric pyridoxal 5'-phosphate (PLP)-dependent enzyme that also catalyzes the physiologically relevant β-elimination of both L- and D-serine to pyruvate and ammonia. After improving the protein purification yield and stability, which had so far limited the biochemical characterization of hSR, we found that the catalytic activity is affected by halides, in the order fluoride > chloride > bromide. On the contrary, iodide elicited a complete inhibition, accompanied by a modulation of the tautomeric equilibrium of the internal aldimine. We also investigated the reciprocal effects of ATP and malonate, an inhibitor that reversibly binds at the active site, 20 Å away from the ATP-binding site. ATP increased ninefold the affinity of hSR for malonate and malonate increased 100-fold that of ATP, confirming an allosteric interaction between the two binding sites. To further investigate this allosteric communication, we probed the active site accessibility by quenching of the coenzyme fluorescence in the absence and presence of ATP. We found that ATP stabilizes a closed conformation of the external aldimine Schiff base, suggesting a possible mechanism for ATP-induced hSR activation.

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pH Dependence of Tryptophan Synthase Catalytic Mechanism

Cited by 10 publications

References 62 publications

Tryptophan synthase: a mine for enzymologists

Tryptophan synthase: a mine for enzymologists

Exploring the pyridoxal 5′‐phosphate‐dependent enzymes

Regulation of human serine racemase activity and dynamics by halides, ATP and malonate

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