Mechanistic deductions from multiple kinetic and solvent deuterium isotope effects and pH studies of pyridoxal phosphate dependent carbon-carbon lyases: Escherichia coli tryptophan indole-lyase

Kiick, Dennis M.; Phillips, Robert S.

doi:10.1021/bi00419a024

Cited by 58 publications

(53 citation statements)

References 21 publications

(14 reference statements)

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“…Similar mechanisms are utilized by other PLP enzymes that catalyze ␤-elimination and ␤-replacement reactions (28,31), including O-acetylserine sulfhydrylase (32,33), tryptophan indole lyase (34), and tyrosine phenol lyase (35). The reactions in Scheme I include the release and transfer of three different protons.…”

Section: Discussionmentioning

confidence: 92%

Catalytic Mechanism of the Tryptophan Synthase α2β2 Complex

Ro¹,

Miles

1999

Journal of Biological Chemistry

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The mechanism of the tryptophan synthase ␣ 2 ␤ 2 complex from Salmonella typhimurium is explored by determining the effects of pH, of temperature, and of isotopic substitution on the pyridoxal phosphate-dependent reaction of L-serine with indole to form L-tryptophan. The pH dependence of the kinetic parameters indicates that three ionizing groups are involved in substrate binding and catalysis with pK a 1 ‫؍‬ 6.5, pK a 2 ‫؍‬ 7.3, and pK a 3 ‫؍‬ 8.2-9. A significant primary isotope effect (ϳ3.5) on V and V/K is observed at low pH (pH 7), but not at high pH (pH 9), indicating that the base that accepts the ␣-proton (␤Lys-87) is protonated at low pH, slowing the abstraction of the ␣-proton and making this step at least partially rate-limiting. pK a 2 is assigned to ␤Lys-87 on the basis of the kinetic isotope effect results and of the observation that the competitive inhibitors glycine and oxindolyl-L-alanine display single pK i values of 7.3. The residue with this pK a (␤Lys-87) must be unprotonated for binding glycine or oxindolyl-L-alanine, and, by inference, L-serine. Investigations of the temperature dependence of the pK a values support the assignment of pK a 2 to ␤Lys-87 and suggest that the ionizing residue with pK a 1 could be a carboxylate, possibly ␤Asp-305, and that the residue associated with a conformational change at pK a 3 may be ␤Lys-167. The occurrence of a closed to open conformational conversion at high pH is supported by investigations of the effects of pH on reaction specificity and on the equilibrium distribution of enzyme-substrate intermediates.The tryptophan synthase ␣ 2 ␤ 2 complex (EC 4.1.2.20) is a useful system for investigating relationships between protein structure and function (for reviews, see Refs. 1-4). The separate tryptophan synthase ␤ 2 subunit 1 and the ␤ subunit in the ␣ 2 ␤ 2 complex catalyze the pyridoxal phosphate (PLP) 2 -dependent conversion of L-serine and indole to L-tryptophan. A number of intermediates in this reaction have been identified from spectroscopic and kinetic studies (see Scheme I in Discussion). The three-dimensional structure of the tryptophan synthase ␣ 2 ␤ 2 complex from Salmonella typhimurium (5) revealed that the active sites of the ␣ and ␤ subunits are ϳ25 Å apart and are connected by a hydrophobic tunnel. This tunnel serves as a passageway for indole from the active site of the ␣ subunit, where it is produced by cleavage of indole-3-glycerol phosphate, to the active site of the ␤ subunit, where it reacts with L-serine to form L-tryptophan. The activity at each site is modulated by reciprocal communication between the ␣ and ␤ sites. These heterotrophic interactions are proposed to switch the ␣ and ␤ subunits between "open" (catalytically inactive) and "closed" (catalytically active) conformations, to coordinate the activities at the two sites, and to prevent the escape of indole (4). Recent crystallographic results provide direct evidence for ligand-induced conformational changes that result in physical closure of loop structures in the ␣ subunit a...

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Section: Discussionmentioning

confidence: 92%

Catalytic Mechanism of the Tryptophan Synthase α2β2 Complex

Ro¹,

Miles

1999

Journal of Biological Chemistry

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“…The elimination of the aromatic leaving group takes place concomitant with or after tautomerization of the initial quinonoid intermediate (31). The strict reaction specificity of these enzymes has been proposed to be controlled by the position of a second catalytically essential base (shown as B 2 in Scheme 3) in the substrate binding site, which controls the tautomerization (3)(4)(5). Structural studies and kinetic analyses of R381A, R381I, and R381V TPL previously demonstrated that Arg-381 is required for the tyrosine substrate specificity of TPL (15), and thus it may be the proposed catalytic base.…”

Section: Discussionmentioning

confidence: 99%

“…Indeed, only one structure has been reported for a quinonoid intermediate in a PLP-dependent enzyme to date (19), and the structure of the quinonoid complex formed in the reaction of indoline and L-Ser catalyzed by tryptophan synthase is in progress. 4 …”

Section: Discussionmentioning

confidence: 99%

“…The amino acid sequence alignment and crystallographic structures show that these two enzymes are very closely related (1,2). Mechanistic studies (3)(4)(5) demonstrated that TPL and Trpase follow very similar catalytic mechanisms (Scheme 3). Both enzymes can catalyze the elimination reactions in vitro of a wide range of amino acids with suitable leaving groups on the ␤-carbon including S-(o-nitrophenyl)-L-cysteine (6,7), S-alkyl-L-cysteines (8,9), ␤-chloro-L-alanine (9, 10), L-Ser (8), and Oacyl-L-serines (7).…”

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confidence: 94%

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Crystals of Tryptophan Indole-Lyase and Tyrosine Phenol-Lyase Form Stable Quinonoid Complexes

Phillips

Demidkina

Zakomirdina

et al. 2002

Journal of Biological Chemistry

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The binding of substrates and inhibitors to wild-typeTyrosine phenol-lyase (TPL) 1 (EC 4.1.99.2) and tryptophan indole-lyase (Trpase, EC 4.1.99.1) are pyridoxal 5Ј-phosphate (PLP)-dependent enzymes that catalyze ␤-elimination reactions to form phenol or indole and ammonium pyruvate from tyrosine and tryptophan, respectively (Schemes 1 and 2) (1, 2). The amino acid sequence alignment and crystallographic structures show that these two enzymes are very closely related (1, 2). Mechanistic studies (3-5) demonstrated that TPL and Trpase follow very similar catalytic mechanisms (Scheme 3). Both enzymes can catalyze the elimination reactions in vitro of a wide range of amino acids with suitable leaving groups on the (8), and Oacyl-L-serines (7). However, in vivo, the enzymes are extremely specific for their respective physiological substrates. Both TPL and Trpase react with substrates and inhibitors to form equilibrating mixtures of external aldimine and quinonoid complexes (5, 10 -14). We have previously reported the x-ray crystallographic structure of TPL complexed with the substrate analog 3-(4Ј-hydroxyphenyl)propionic acid (15). This complex resembles the Michaelis-Menten complex, because the analog lacks an amino group and is unable to form an external aldimine. We would like to obtain structures of quinonoid intermediates for both enzymes, because quinonoid intermediates are proposed to play a central role in the mechanisms of both enzymes (Scheme 3). In previous studies with Trpase and tryptophan synthase (16, 17), we prepared inhibitors oxindolyl-Lalanine (Scheme 4, I) and dihydro-L-tryptophan, which resemble the indolenine intermediate (Scheme 3), a proposed intermediate in the reactions of both enzymes. Trpase and tryptophan synthase are inhibited by different diastereomers of dihydro-L-tryptophan, suggesting that the indolenine intermediates in the reactions of the two enzymes exhibit opposite chirality (17). Oxindolyl-L-alanine was found previously to inhibit Escherichia coli Trpase with a K i value of ϳ2.5-6 M (3, 16, 18) and to form a prominent absorption band at 502 nm, demonstrating the predominant formation of a stable quinon-

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“…Moreover, the above multistep reaction involving hydrogen transfer is still not clear. A number of mechanistic questions can be resolved [4][5][6] by determining kinetic isotope effects, KIE, using tritium attached to selected positions of l-Trp and 5-OH-l-Trp. The numerical values of the KIE may be useful in distinguishing between alternative mechanisms.…”

Section: Introductionmentioning

confidence: 99%

Synthesis of [¹⁴C]‐l‐tryptophan and [¹⁴C]‐5′‐hydroxy‐l‐tryptophan labeled in the carboxyl group

Boroda

Kański

Kańska

2003

Labelled Comp Radiopharmac

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The synthesis of selectively 14C‐labeled l‐tryptophan and its derivative 5‐hydroxy‐l‐tryptophan using chemical and multienzymatic methods is reported. The mixture containing [1‐14C[‐dl‐alanine, indole or 5‐hydroxyindole has been converted to [1‐14C]‐l‐tryptophan or 5′‐hydroxy‐[1‐14C]‐l‐tryptophan, respectively, in a one‐pot multienzymatic reaction using four enzymes: d‐amino acid oxidase, catalase, glutamic‐pyruvic transaminase and tryptophanase. Copyright © 2003 John Wiley & Sons, Ltd.

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Mechanistic deductions from multiple kinetic and solvent deuterium isotope effects and pH studies of pyridoxal phosphate dependent carbon-carbon lyases: Escherichia coli tryptophan indole-lyase

Cited by 58 publications

References 21 publications

Catalytic Mechanism of the Tryptophan Synthase α2β2 Complex

Catalytic Mechanism of the Tryptophan Synthase α2β2 Complex

Crystals of Tryptophan Indole-Lyase and Tyrosine Phenol-Lyase Form Stable Quinonoid Complexes

Synthesis of [¹⁴C]‐l‐tryptophan and [¹⁴C]‐5′‐hydroxy‐l‐tryptophan labeled in the carboxyl group

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Mechanistic deductions from multiple kinetic and solvent deuterium isotope effects and pH studies of pyridoxal phosphate dependent carbon-carbon lyases: Escherichia coli tryptophan indole-lyase

Cited by 58 publications

References 21 publications

Catalytic Mechanism of the Tryptophan Synthase α2β2 Complex

Catalytic Mechanism of the Tryptophan Synthase α2β2 Complex

Crystals of Tryptophan Indole-Lyase and Tyrosine Phenol-Lyase Form Stable Quinonoid Complexes

Synthesis of [14C]‐l‐tryptophan and [14C]‐5′‐hydroxy‐l‐tryptophan labeled in the carboxyl group

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Synthesis of [¹⁴C]‐l‐tryptophan and [¹⁴C]‐5′‐hydroxy‐l‐tryptophan labeled in the carboxyl group