Interactions of tryptophan synthase, tryptophanase, and pyridoxal phosphate with oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan: support for an indolenine intermediate in tryptophan metabolism

Phillips, Robert S.; Miles, Edith Wilson; Cohen, Louis A.

doi:10.1021/bi00320a052

Cited by 81 publications

(88 citation statements)

References 41 publications

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“…The rates of indole channeling and the reaction of indole with the aminoacrylate to form tryptophan are set at the lower limit sufficient to account for the accumulation of indole in the single turnover experiments. In fitting the data, the rate constants for the reaction were further constrained by dissociation constants and K m values for IGP, G3P, serine, and tryptophan as previously determined Kawasaki et al, 1987;Phillips et al, 1984) and in Table I in this work.…”

Section: Resultsmentioning

confidence: 99%

“…The entire data set was fit to a single model and a single set of rate constants. The data were fit by a trial and error process maintaining the constraints of known dissociation constants and K m values for IGP, G3P, indole, tryptophan, and serine Kawasaki et al, 1987;Phillips et al, 1984). Not all of the rate constants are known with equal certainty.…”

Section: Methodsmentioning

confidence: 99%

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Kinetic Characterization of Channel Impaired Mutants of Tryptophan Synthase

Anderson

Kim

Quillen

et al. 1995

Journal of Biological Chemistry

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Tryptophan synthase, an ␣ 2 ␤ 2 tetrameric complex, is a classic example of an enzyme that is thought to "channel" a metabolic intermediate (indole) from the active site of the ␣ subunit to the active site of the ␤ subunit. The solution of the three-dimensional structure of the enzyme from Salmonella typhimurium provided physical evidence for a 25-Å hydrophobic tunnel which connects the ␣ and ␤ active sites (Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., and Davies, D. R. (1988) J. Biol. Chem. 263, 17857-17871). Using rapid reaction kinetics, we have previously established that indole is indeed channeled and have identified three essential kinetic features which govern efficient channeling. In the current study we have probed the necessity of these features by using site-directed mutagenesis to alter these requirements. We now report the kinetic characterization of two mutants which contain substitutions to block or restrict the tunnel (␤C170F and ␤C170W). Preliminary kinetic and structural evidence of a restricted tunnel in the ␤C170W has been provided (Schlichting, I., Yang, X. W., Miles, E. W., Kim, A. Y., and Anderson, K. S. (1994) J. Biol. Chem. 269, 26591-26593). The rapid kinetic analysis of these mutant proteins shows that these mutations interfere with efficient channeling of the indole metabolite such that indole can be observed in single enzyme turnover of the physiologically relevant ␣␤ reaction. In addition, the ␤C170W mutant appears to be impaired in ␣␤ intersubunit communication.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Kinetic Characterization of Channel Impaired Mutants of Tryptophan Synthase

Anderson

Kim

Quillen

et al. 1995

Journal of Biological Chemistry

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“…Oxindolyl-L-alanine, which has tetrahedral geometry at C-3 of the indole ring, is a structural analog of the indolenine tautomer of L-tryptophan, which is a proposed intermediate in reactions of tryptophan synthase (E-V in Scheme I) and tryptophan indole-lyase (13,29). The finding that oxindolyl-L-alanine is a potent competitive inhibitor of both enzymes provided strong evidence for the intermediacy of the indolenine tautomer of L-tryptophan in reactions catalyzed (13,29).…”

Section: Discussionmentioning

confidence: 99%

“…Oxindolyl-Lalanine, which was a generous gift of Robert S. Phillips, was synthesized by the method of Savige and Fontana (12) as described (13). MOPS and proline were from Fluka.…”

Section: Methodsmentioning

confidence: 99%

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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“…The most efficient inhibitor of Tnase known hitherto, oxindolyl-L-alanine, (Ki ¼ 5mM) inhibits both the formation of indole and the creation of biofilm [20,11]. Other quasi-substrates of Tnase such as alanine and phenylalanine inhibit the enzyme in the mM concentrations range [21].…”

Section: Introductionmentioning

confidence: 99%

New tryptophanase inhibitors: Towards prevention of bacterial biofilm formation

Scherzer

Gdalevsky

Goldgur

et al. 2008

Journal of Enzyme Inhibition and Medicinal Chemistry

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Tryptophanase (tryptophan indole-lyase, Tnase, EC 4.1.99.1), a bacterial enzyme with no counterpart in eukaryotic cells, produces from L-tryptophan pyruvate, ammonia and indole. It was recently suggested that indole signaling plays an important role in the stable maintenance of multicopy plasmids. In addition, Tnase was shown to be capable of binding Rcd, a short RNA molecule involved in resolution of plasmid multimers. Binding of Rcd increases the affinity of Tnase for tryptophan, and it was proposed that indole is involved in bacteria multiplication and biofilm formation. Biofilm-associated bacteria may cause serious infections, and biofilm contamination of equipment and food, may result in expensive consequences. Thus, optimal and specific factors that interact with Tnase can be used as a tool to study the role of this multifunctional enzyme as well as antibacterial agents that may affect biofilm formation. Most known quasi-substrates inhibit Tnase at the mM range. In the present work, the mode of Tnase inhibition by the following compounds and the corresponding Ki values were: S-phenylbenzoquinone-L-tryptophan, uncompetitively, 101 mM; a-amino-2-(9,10-anthraquinone)-propanoic acid, noncompetitively, 174 mM; L-tryptophane-ethylester, competitively, 52 mM; N-acetyl-L-tryptophan, noncompetitively, 48 mM. S-phenylbenzoquinone-L-tryptophan and a-amino-2-(9,10-anthraquinone)-propanoic acid were newly synthesized.

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Interactions of tryptophan synthase, tryptophanase, and pyridoxal phosphate with oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan: support for an indolenine intermediate in tryptophan metabolism

Cited by 81 publications

References 41 publications

Kinetic Characterization of Channel Impaired Mutants of Tryptophan Synthase

Kinetic Characterization of Channel Impaired Mutants of Tryptophan Synthase

Catalytic Mechanism of the Tryptophan Synthase α2β2 Complex

New tryptophanase inhibitors: Towards prevention of bacterial biofilm formation

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