RESUMEN
Antibiotic resistance is a continually growing challenge in the treatment of various bacterial infections worldwide. New drugs and new drug targets are necessary to curb the threat of infectious diseases caused by multidrug-resistant pathogens. The tryptophan biosynthesis pathway is essential for bacterial growth but is absent in higher animals and humans. Drugs that can inhibit the bacterial biosynthesis of tryptophan offer a new class of antibiotics. In this work, we combined a structure-based strategy using in silico docking screening and molecular dynamics (MD) simulations to identify compounds targeting the α subunit of tryptophan synthase with experimental methods involving the whole-cell minimum inhibitory concentration (MIC) test, solution state NMR, and crystallography to confirm the inhibition of L-tryptophan biosynthesis. Screening 1,800 compounds from the National Cancer Institute Diversity Set I against α subunit revealed 28 compounds for experimental validation; four of the 28 hit compounds showed promising activity in MIC testing. We performed solution state NMR experiments to demonstrate that a one successful inhibitor, 3-amino-3-imino-2-phenyldiazenylpropanamide (Compound 1) binds to the α subunit. We also report a crystal structure of Salmonella enterica serotype Typhimurium tryptophan synthase in complex with Compound 1 which revealed a binding site at the αß interface of the dimeric enzyme. MD simulations were carried out to examine two binding sites for the compound. Our results show that this small molecule inhibitor could be a promising lead for future drug development.
Asunto(s)
Antibacterianos , Triptófano Sintasa , Antibacterianos/química , Antibacterianos/farmacología , Sitios de Unión , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Triptófano Sintasa/antagonistas & inhibidores , Triptófano Sintasa/químicaRESUMEN
We have examined the reaction of Salmonella enterica serovar typhimurium tryptophan (Trp) synthase α2ß2 complex with l-Trp, d-Trp, oxindolyl-l-alanine (OIA), and dioxindolyl-l-alanine (DOA) in the presence of disodium (dl)-α-glycerol phosphate (GP), using stopped-flow spectrophotometry and X-ray crystallography. All structures contained the d-isomer of GP bound at the α-active site. (3S)-OIA reacts with the pyridoxal-5'-phosphate (PLP) of Trp synthase to form a mixture of external aldimine and quinonoid complexes. The α-carboxylate of OIA rotates about 90° to become planar with the PLP when the quinonoid complex is formed, resulting in a conformational change in the loop of residues 110-115. The COMM domain of the Trp synthase-OIA complex is found as a mixture of two conformations. The (3R)-diastereomer of DOA binds about 5-fold more tightly than (3S)-OIA and also forms a mixture of aldimine and quinonoid complexes. DOA forms an additional H-bond between the 3-OH of DOA and ßLys-87. l-Trp does not form a covalent complex with the PLP of Trp synthase. However, d-Trp forms a mixture of two external aldimine complexes which differ in the orientation of the α-carboxylate. In one conformation, the α-carboxylate is in the plane of the PLP, while in the other conformation, the α-carboxylate is perpendicular to the PLP plane. These results confirm that the stereochemistry of the transient indolenine quinonoid intermediate in the mechanism of Trp synthase is (3S) and demonstrate the linkage between aldimine and quinonoid reaction intermediates in the ß-active site and allosteric communications with the α-active site.
Asunto(s)
Proteínas Bacterianas/química , Salmonella typhimurium/enzimología , Triptófano Sintasa/química , Triptófano/química , Proteínas Bacterianas/antagonistas & inhibidores , Dominio Catalítico , Estructura Secundaria de Proteína , Triptófano Sintasa/antagonistas & inhibidoresRESUMEN
Light regulation of drug molecules has gained growing interest in biochemical and pharmacological research in recent years. In addition, a serious need for novel molecular targets of antibiotics has emerged presently. Herein, the development of a photocontrollable, azobenzene-based antibiotic precursor towards tryptophan synthase (TS), an essential metabolic multienzyme complex in bacteria, is presented. The compound exhibited moderately strong inhibition of TS in its E configuration and five times lower inhibition strength in its Z configuration. A combination of biochemical, crystallographic, and computational analyses was used to characterize the inhibition mode of this compound. Remarkably, binding of the inhibitor to a hitherto-unconsidered cavity results in an unproductive conformation of TS leading to noncompetitive inhibition of tryptophan production. In conclusion, we created a promising lead compound for combatting bacterial diseases, which targets an essential metabolic enzyme, and whose inhibition strength can be controlled with light.
Asunto(s)
Compuestos Azo/farmacología , Inhibidores Enzimáticos/farmacología , Triptófano Sintasa/antagonistas & inhibidores , Inhibidores Enzimáticos/efectos de la radiaciónRESUMEN
Global dispersion of multidrug resistant bacteria is very common and evolution of antibiotic-resistance is occurring at an alarming rate, presenting a formidable challenge for humanity. The development of new therapeuthics with novel molecular targets is urgently needed. Current drugs primarily affect protein, nucleic acid, and cell wall synthesis. Metabolic pathways, including those involved in amino acid biosynthesis, have recently sparked interest in the drug discovery community as potential reservoirs of such novel targets. Tryptophan biosynthesis, utilized by bacteria but absent in humans, represents one of the currently studied processes with a therapeutic focus. It has been shown that tryptophan synthase (TrpAB) is required for survival of Mycobacterium tuberculosis in macrophages and for evading host defense, and therefore is a promising drug target. Here we present crystal structures of TrpAB with two allosteric inhibitors of M. tuberculosis tryptophan synthase that belong to sulfolane and indole-5-sulfonamide chemical scaffolds. We compare our results with previously reported structural and biochemical studies of another, azetidine-containing M. tuberculosis tryptophan synthase inhibitor. This work shows how structurally distinct ligands can occupy the same allosteric site and make specific interactions. It also highlights the potential benefit of targeting more variable allosteric sites of important metabolic enzymes.
Asunto(s)
Sitio Alostérico/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Indoles/farmacología , Mycobacterium tuberculosis/enzimología , Sulfonamidas/farmacología , Tiofenos/farmacología , Triptófano Sintasa/antagonistas & inhibidores , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Humanos , Indoles/química , Ligandos , Modelos Moleculares , Estructura Molecular , Sulfonamidas/química , Tiofenos/química , Triptófano Sintasa/química , Triptófano Sintasa/metabolismoRESUMEN
Multi-drug-resistant tuberculosis and extensively drug-resistant tuberculosis has emerged as global health threat, causing millions of deaths worldwide. Identification of new drug candidates for tuberculosis (TB) by targeting novel and less explored protein targets will be invaluable for antituberculosis drug discovery. We performed structure-based virtual screening of eMolecules database against a homology model of relatively unexplored protein target: the α-subunit of tryptophan synthase (α-TRPS) from Mycobacterium tuberculosis essential for bacterial survival. Based on physiochemical properties analysis and molecular docking, the seven candidate compounds were selected and evaluated through whole cell-based activity against the H37Rv strain of M. tuberculosis. A new Benzamide inhibitor against α-subunit of tryptophan synthase (α-TRPS) from M. tuberculosis has been identified causing 100% growth inhibition at 25 µg/ml and visible bactericidal activity at 6 µg/ml. This benzamide inhibitor displayed a good predicted binding score (-48.24 kcal/mol) with the α-TRPS binding pocket and has logP value (2.95) comparable to Rifampicin. Further refinement of docking results and evaluation of inhibitor-protein complex stability were investigated through Molecular dynamic (MD) simulations studies. Following MD simulations, Root mean square deviation, Root mean square fluctuation and secondary structure analysis confirmed that protein did not unfold and ligand stayed inside the active pocket of protein during the explored time scale. This identified benzamide inhibitor against the α-subunit of TRPS from M. tuberculosis could be considered as candidate for drug discovery against TB and will be further evaluated for enzyme-based inhibition in future studies.
Asunto(s)
Antituberculosos/farmacología , Benzamidas/química , Ensayos Analíticos de Alto Rendimiento/métodos , Simulación de Dinámica Molecular , Mycobacterium tuberculosis/enzimología , Triptófano Sintasa/antagonistas & inhibidores , Tuberculosis/tratamiento farmacológico , Antituberculosos/química , Proteínas Bacterianas/antagonistas & inhibidores , Bases de Datos Farmacéuticas , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Mycobacterium tuberculosis/efectos de los fármacos , Conformación Proteica , Subunidades de Proteína , Tuberculosis/microbiologíaRESUMEN
Drug discovery efforts against the pathogen Mycobacterium tuberculosis (Mtb) have been advanced through phenotypic screens of extensive compound libraries. Such a screen revealed sulfolane 1 and indoline-5-sulfonamides 2 and 3 as potent inhibitors of mycobacterial growth. Optimization in the sulfolane series led to compound 4, which has proven activity in an in vivo murine model of Mtb infection. Here we identify the target and mode of inhibition of these compounds based on whole genome sequencing of spontaneous resistant mutants, which identified mutations locating to the essential α- and ß-subunits of tryptophan synthase. Over-expression studies confirmed tryptophan synthase as the biological target. Biochemical techniques probed the mechanism of inhibition, revealing the mutant enzyme complex incurs a fitness cost but does not prevent inhibitor binding. Mapping of the resistance conferring mutations onto a low-resolution crystal structure of Mtb tryptophan synthase showed they locate to the interface between the α- and ß-subunits. The discovery of anti-tubercular agents inhibiting tryptophan synthase highlights the therapeutic potential of this enzyme and draws attention to the prospect of other amino acid biosynthetic pathways as future Mtb drug targets.
Asunto(s)
Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Mycobacterium/efectos de los fármacos , Mycobacterium/enzimología , Triptófano Sintasa/antagonistas & inhibidores , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana , Humanos , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Mutación , Mycobacterium/genética , Conformación Proteica , Relación Estructura-Actividad , Tiofenos/farmacología , Triptófano Sintasa/química , Triptófano Sintasa/metabolismoRESUMEN
New antibiotics with novel targets are greatly needed. Bacteria have numerous essential functions, but only a small fraction of such processes-primarily those involved in macromolecular synthesis-are inhibited by current drugs. Targeting metabolic enzymes has been the focus of recent interest, but effective inhibitors have been difficult to identify. We describe a synthetic azetidine derivative, BRD4592, that kills Mycobacterium tuberculosis (Mtb) through allosteric inhibition of tryptophan synthase (TrpAB), a previously untargeted, highly allosterically regulated enzyme. BRD4592 binds at the TrpAB α-ß-subunit interface and affects multiple steps in the enzyme's overall reaction, resulting in inhibition not easily overcome by changes in metabolic environment. We show that TrpAB is required for the survival of Mtb and Mycobacterium marinum in vivo and that this requirement may be independent of an adaptive immune response. This work highlights the effectiveness of allosteric inhibition for targeting proteins that are naturally highly dynamic and that are essential in vivo, despite their apparent dispensability under in vitro conditions, and suggests a framework for the discovery of a next generation of allosteric inhibitors.
Asunto(s)
Antituberculosos , Azetidinas/química , Mycobacterium tuberculosis/enzimología , Bibliotecas de Moléculas Pequeñas , Triptófano Sintasa/antagonistas & inhibidores , Regulación Alostérica , Antituberculosos/síntesis química , Antituberculosos/química , Antituberculosos/farmacología , Azetidinas/farmacología , Sitios de Unión , Cristalografía por Rayos X , Sistemas de Liberación de Medicamentos , Mycobacterium tuberculosis/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
The development of new therapies against infectious diseases is vital in developing countries. Among infectious diseases, tuberculosis is considered the leading cause of death. A target for development of new drugs is the tryptophan pathway. The last enzyme of this pathway, tryptophan synthase (TRPS), is responsible for conversion of the indole 3-glycerol phosphate into indol and the condensation of this molecule with serine-producing tryptophan. The present work describes the molecular models of TRPS from Mycobacterium tuberculosis (MtTRPS) complexed with six inhibitors, the indole 3-propanol phosphate and five arylthioalkyl-phosphonated analogs of substrate of the alpha-subunit. The molecular models of MtTRPS present good stereochemistry, and the binding of the inhibitors is favorable. Thus, the generated models can be used in the design of more specific drugs against tuberculosis and other infectious diseases.
Asunto(s)
Modelos Moleculares , Mycobacterium tuberculosis/enzimología , Triptófano Sintasa/antagonistas & inhibidores , Triptófano Sintasa/química , Sitios de Unión , Simulación por Computador , Diseño de Fármacos , Fagaceae/genética , Enlace de Hidrógeno , Ligandos , Estructura Molecular , Proteínas de Plantas/química , Proteínas de Plantas/clasificación , Unión Proteica/fisiología , Conformación Proteica , Rosaceae/genética , Alineación de Secuencia , Especificidad por SustratoRESUMEN
Some non-detergent sulfobetaines had been shown to prevent aggregation and improve the yield of active proteins when added to the buffer during in vitro protein renaturation. With the aim of designing more efficient folding helpers, a series of non-detergent sulfobetaines have been synthesized and their efficiency in improving the renaturation of a variety of proteins (E. coli tryptophan synthase and beta-D-galactosidase, hen lysozyme, bovine serum albumin, a monoclonal antibody) have been investigated. Attempts to correlate the structure of each sulfobetaines with its effect on folding revealed some molecular features that appear important in helping renaturation. This enabled us to design and synthesize new non-detergent sulfobetaines that act as potent folding helpers.
Asunto(s)
Betaína/análogos & derivados , Pliegue de Proteína , Animales , Anticuerpos Bloqueadores/química , Anticuerpos Monoclonales/química , Betaína/química , Bovinos , Fenómenos Químicos , Química Física , Pollos , Diseño de Fármacos , Ratones , Muramidasa/química , Renaturación de Proteína , Proteínas/química , Albúmina Sérica Bovina/química , Triptófano Sintasa/antagonistas & inhibidores , Triptófano Sintasa/químicaRESUMEN
Substrate channeling in the tryptophan synthase bienzyme is regulated by allosteric interactions. Allosteric signals are transmitted via a scaffolding of structural elements that includes a monovalent cation-binding site and salt-bridging interactions between the side chains of betaAsp 305, betaArg 141, betaLys 167, and alphaAsp 56 that appear to modulate the interconversion between open and closed conformations. betaAsp 305 also interacts with the hydroxyl group of the substrate L-Ser in some structures. One possible functional role for betaAsp 305 is to ensure the allosteric transmission that triggers the switching of alphabeta-dimeric units between open and closed conformations of low and high activity. This work shows that substitution of betaAsp 305 with Ala (betaD305A) decreases the affinity of the beta-site for the substrate L-Ser, destabilizes the enzyme-bound alpha-aminoacrylate, E(A-A), and quinonoid species, E(Q), and changes the nucleophile specificity of the beta-reaction. The altered specificity provides a biosynthetic route for new L-amino acids derived from substrate analogues. betaD305A also shows an increased rate of formation of pyruvate upon reaction with L-Ser relative to the wild-type enzyme. The formation of pyruvate is strongly inhibited by the binding of benzimidazole to E(A-A). Upon reaction with L-Ser and in the presence of the alpha-site substrate analogue, alpha-glycerol phosphate, the Na(+) form of betaD305A undergoes inactivation via reaction of nascent alpha-aminoacrylate with bound PLP. This work establishes important roles for betaAsp 305 both in the conformational change between open and closed states that takes place at the beta-site during the formation of the E(A-A) and in substrate binding and recognition.
Asunto(s)
Alanina/genética , Ácido Aspártico/genética , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Mutagénesis Sitio-Dirigida , Triptófano Sintasa/genética , Triptófano Sintasa/metabolismo , beta-Alanina/análogos & derivados , Regulación Alostérica/genética , Compuestos de Anilina/metabolismo , Bencimidazoles/metabolismo , Catálisis , Deuterio/metabolismo , Activación Enzimática/genética , Hidroxilaminas/metabolismo , Indoles/metabolismo , Cinética , Complejos Multienzimáticos/antagonistas & inhibidores , Ácido Pirúvico/metabolismo , Salmonella typhimurium/enzimología , Salmonella typhimurium/genética , Serina/análogos & derivados , Serina/metabolismo , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , Especificidad por Sustrato/genética , Volumetría , Triptófano Sintasa/antagonistas & inhibidores , beta-Alanina/metabolismoRESUMEN
In an effort to use a structure-based approach for the design of new herbicides, the crystal structures of complexes of tryptophan synthase with a series of phosphonate enzyme inhibitors were determined at 2.3 A or higher resolution. These inhibitors were designed to mimic the transition state formed during the alpha-reaction of the enzyme and, as expected, have affinities much greater than that of the natural substrate indole-3-glycerol phosphate or its nonhydrolyzable analogue indole propanol phosphate (IPP). These inhibitors are ortho-substituted arylthioalkylphosphonate derivatives that have an sp(3)-hybridized sulfur atom, designed to mimic the putative tetrahedral transition state at the C3 atom of the indole, and lack the C2 atom to allow for higher conformational flexibility. Overall, the inhibitors bind in a fashion similar to that of IPP. Glu-49 and Phe-212 are the two active site residues whose conformation changes upon inhibitor binding. A very short hydrogen bond between a phosphonate oxygen and the Ser-235 hydroxyl oxygen may be responsible for stabilization of the enzyme-inhibitor complexes. Implications for the mechanism of catalysis as well as directions for more potent inhibitors are discussed.
Asunto(s)
Organofosfonatos/química , Triptófano Sintasa/química , Cristalografía por Rayos X , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Enlace de Hidrógeno , Modelos Moleculares , Triptófano Sintasa/antagonistas & inhibidoresRESUMEN
The mechanism of the tryptophan synthase alpha(2)beta(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 (approximately 3.5) 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 alpha-proton (betaLys-87) is protonated at low pH, slowing the abstraction of the alpha-proton and making this step at least partially rate-limiting. pK(a)2 is assigned to betaLys-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) (betaLys-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 betaLys-87 and suggest that the ionizing residue with pK(a)1 could be a carboxylate, possibly betaAsp-305, and that the residue associated with a conformational change at pK(a)3 may be betaLys-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.
Asunto(s)
Salmonella typhimurium/enzimología , Triptófano Sintasa/metabolismo , Alanina/análogos & derivados , Alanina/farmacología , Regulación Alostérica , Dominio Catalítico , Deuterio , Glicina/farmacología , Concentración de Iones de Hidrógeno , Indoles/metabolismo , Ligandos , Modelos Químicos , Oxindoles , Fosfato de Piridoxal , Serina/metabolismo , Triptófano Sintasa/antagonistas & inhibidoresRESUMEN
Compounds designed to mimic the tryptophan synthase alpha subunit reactive intermediate were found to be potent inhibitors of the enzyme. These compounds are herbicidal and the herbicidal mode of action was demonstrated to be due to disruption of tryptophan biosynthesis.
Asunto(s)
Herbicidas/síntesis química , Triptófano/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Herbicidas/farmacología , Triptófano/biosíntesis , Triptófano Sintasa/antagonistas & inhibidoresRESUMEN
Mutations in the pyridoxal phosphate binding site of the tryptophan synthase beta subunit (S377D and S377E) alter cofactor chemistry [Jhee, K.-H., et al. (1998) J. Biol. Chem. 273, 11417-11422]. We now report that the S377D, S377E, and S377A beta2 subunits form alpha2 beta2 complexes with the alpha subunit and activate the alpha subunit-catalyzed cleavage of indole 3-glycerol phosphate. The apparent Kd for dissociation of the alpha and beta subunits is unaffected by the S377A mutation but is increased up to 500-fold by the S377D and S377E mutations. Although the three mutant alpha2 beta2 complexes exhibit very low activities in beta elimination and beta replacement reactions catalyzed at the beta site in the presence of Na+, the activities and spectroscopic properties of the S377A alpha2 beta2 complex are partially repaired by addition of Cs+. The S377D and S377E alpha2 beta2 complexes, unlike the wild-type and S377A alpha2 beta2 complexes and the mutant beta2 subunits, undergo irreversible substrate-induced inactivation by L-serine or by beta-chloro-L-alanine. The rates of inactivation (kinact) are similar to the rates of catalysis (kcat). The partition ratios are very low (kcat/kinact = 0.25-3) and are affected by alpha subunit ligands and monovalent cations. The inactivation product released by alkali was shown by HPLC and by fluorescence, absorption, and mass spectroscopy to be identical to a compound previously synthesized from pyridoxal phosphate and pyruvate. We suggest that alterations in the cofactor chemistry that result from the engineered Asp377 in the active site of the beta subunit may promote the mechanism-based inactivation.
Asunto(s)
Mutagénesis Sitio-Dirigida , Fosfato de Piridoxal/química , Triptófano Sintasa/genética , Alanina/genética , Ácido Aspártico/genética , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Catálisis , Dicroismo Circular , Activación Enzimática/genética , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosfato de Piridoxal/metabolismo , Serina/genética , Serina/metabolismo , Espectrometría de Fluorescencia , Especificidad por Sustrato/genética , Triptófano Sintasa/antagonistas & inhibidores , Triptófano Sintasa/químicaRESUMEN
This study investigates effects of ligands on thermal inactivation of the tryptophan synthase alpha and beta 2 subunits alone and in the alpha 2 beta 2 complex. Addition of pyridoxal phosphate to the apo-beta 2 subunit increases the temperature of one-half inactivation (Ti) from 52 to 77 degrees C. Ligands that promote association of the alpha and holo-beta 2 subunits markedly stabilize the more temperature-labile alpha subunit in the alpha 2 beta 2 complex from irreversible thermal denaturation. The combination of a beta 2 subunit ligand (L-serine) with an alpha subunit ligand (alpha-glycerol 3-phosphate) raises the inactivation temperature (Ti) of the alpha subunit in the holo-alpha 2 beta 2 complex from 54 to 66 degrees C. In contrast, values of Ti for inactivation of the alpha and beta subunits in the holo-alpha 2 beta 2 complex are more similar to respective values for the isolated alpha subunit (50 degrees C) and holo-beta 2 subunit (77 degrees C). Surprisingly, the addition of L-serine results in a larger decrease in the Ti of the beta 2 subunit in the holo-alpha 2 beta 2 complex (78 degrees C-->64 degrees C) than in Ti of the holo-beta 2 subunit alone (77 degrees C-->71 degrees C). The observation that ligands have different effects on the isolated and associated subunits provides evidence that the alpha and beta 2 subunits do not fully dissociate during thermal inactivation of the alpha 2 beta 2 complex at pH 7.8 and at approximately 0.1 ionic strength. Our results demonstrate that linkage between protein-ligand interactions and protein-protein interactions affects the conformational stability of the tryptophan synthase alpha 2 beta 2 complex.
Asunto(s)
Salmonella typhimurium/enzimología , Triptófano Sintasa/antagonistas & inhibidores , Triptófano Sintasa/química , Apoenzimas/antagonistas & inhibidores , Apoenzimas/química , Sitios de Unión , Clonación Molecular , Estabilidad de Enzimas , Escherichia coli , Calor , Sustancias Macromoleculares , Fosfato de Piridoxal/farmacología , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Termodinámica , Triptófano Sintasa/biosíntesisRESUMEN
Tryptophan synthase (alpha 2 beta 2 complex) from Salmonella typhimurium catalyzes the formation of tryptophan from serine and indole. The enzyme is inactivated by (1-fluorovinyl)glycine. Concomitant with enzyme inactivation, the absorbance at 485 nm increases, indicating covalent modification of pyridoxal 5'-phosphate. It is proposed that inactivation involves elimination of HF to form an allene, which reacts with a nucleophile at the active site. The inactivation reaction involves an alpha,beta-elimination, as does the formation of tryptophan from indole and serine. The inactivation occurs with k(in) > 1.3 s-1, which is very close to k(cat) (6.4 s-1) for the formation of tryptophan from indole and serine. The inactive enzyme (alpha 2 beta 2) regains activity with k(off) = 0.005 min-1. Aminoacetone is formed during reaction, and pyridoxal 5'-phosphate is regenerated. Tryptophan synthase also catalyzes the dehydration of serine, or 3-fluoroalanine, to pyruvate in the absence of indole. This reaction involves an alpha,beta-elimination and the intermediate formation of an aminoacrylate adduct with pyridoxal 5'-phosphate, as does the formation of tryptophan. Pyruvate formation proceeds at less than 5% the rate of tryptophan formation. With [2-2H]serine an isotope effect (DVmax = 1.5) is observed. We propose that pyruvate formation is limited by the rate of hydration of the aminoacrylate intermediate and the rate of the abstraction of the serine alpha-hydrogen.
Asunto(s)
Glicina/análogos & derivados , Salmonella typhimurium/enzimología , Triptófano Sintasa/antagonistas & inhibidores , Acetona/análogos & derivados , Acetona/metabolismo , Fluoruros/metabolismo , Glicina/farmacología , Cinética , Modelos Químicos , Fosfato de Piridoxal/metabolismo , Espectrofotometría , Triptófano Sintasa/metabolismoRESUMEN
Trifluoroalanine is a mechanism-based inactivator of Escherichia coli tryptophan indole-lyase (tryptophanase) and E. coli tryptophan synthase (R. B. Silverman and R. H. Abeles, 1976, Biochemistry 15, 4718-4723). We have found that indole is able to prevent inactivation of tryptophan indole-lyase by trifluoroalanine. The protection of tryptophan indole-lyase by indole exhibits saturation kinetics, with a KD of 0.03 mM, which is comparable to the KI for inhibition of pyruvate ion formation (0.01 mM) and the Km for L-tryptophan synthesis. Fluoride electrode measurements indicate the formation of 28 mol of fluoride ion per mole of enzyme during inactivation of tryptophan indole-lyase, and 121 mol of fluoride ion are formed per mole of enzyme in the presence of 2 mM indole during the same incubation period. 19F NMR spectra of reaction mixtures of tryptophan indole-lyase and trifluoroalanine showed evidence only for fluoride ion formation, in either the absence or the presence of indole, and difluoropyruvic acid was not detected. The partition ratio, kcat/kinact, is estimated to be 9. Tryptophan indole-lyase in the presence of trifluoroalanine exhibits visible absorption peaks at 446 and 478 nm, which decay at the same rate as inactivation. However, in the presence of 1 mM indole and trifluoralanine, tryptophan indole-lyase exhibits a peak only at 420 nm, and the spectra show a gradual increase at 300-310 nm with incubation. In contrast, tryptophan synthase is not protected by indole from inactivation by trifluoroalanine, and the absorption peak at 408 nm for the tryptophan synthase-trifluoroalanine complex is unaffected by indole. These results demonstrate that inactivation of tryptophan indole-lyase occurs via a catalytically competent species, probably the beta,beta-difluoro-alpha-aminoacrylate intermediate, which can be partitioned from inactivation to products by a reactive aromatic nucleophile, indole.
Asunto(s)
Alanina/análogos & derivados , Escherichia coli/enzimología , Indoles/farmacología , Triptófano Sintasa/antagonistas & inhibidores , Triptofanasa/antagonistas & inhibidores , Alanina/farmacología , Cromatografía Líquida de Alta Presión , Activación Enzimática/efectos de los fármacos , Fluoruros/metabolismo , Cinética , Espectroscopía de Resonancia Magnética , Piruvatos/metabolismo , Ácido Pirúvico , EspectrofotometríaRESUMEN
The origin of reaction and substrate specificity and the control of activity by protein-protein interaction are investigated using the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium. We have compared some spectroscopic and kinetic properties of the wild type beta subunit and five mutant forms of the beta subunit that have altered catalytic properties. These mutant enzymes, which were engineered by site-directed mutagenesis, have single amino acid replacements in either the active site or in the wall of a tunnel that extends from the active site of the alpha subunit to the active site of the beta subunit in the alpha 2 beta 2 complex. We find that the mutant alpha 2 beta 2 complexes have altered reaction and substrate specificity in beta-elimination and beta-replacement reactions with L-serine and with beta-chloro-L-alanine. Moreover, the mutant enzymes, unlike the wild type alpha 2 beta 2 complex, undergo irreversible substrate-induced inactivation. The mechanism of inactivation appears to be analogous to that first demonstrated by Metzler's group for inhibition of two other pyridoxal phosphate enzymes. Alkaline treatment of the inactivated enzyme yields apoenzyme and a previously described pyridoxal phosphate derivative. We demonstrate for the first time that enzymatic activity can be recovered by addition of pyridoxal phosphate following alkaline treatment. We conclude that the wild type and mutant alpha 2 beta 2 complexes differ in the way they process the amino acrylate intermediate. We suggest that the wild type beta subunit undergoes a conformational change upon association with the alpha subunit that alters the reaction specificity and that the mutant beta subunits do not undergo the same conformational change upon subunit association.
Asunto(s)
Mutagénesis Sitio-Dirigida , Salmonella typhimurium/enzimología , Triptófano Sintasa/metabolismo , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Activación Enzimática , Escherichia coli/genética , Cinética , Sustancias Macromoleculares , Modelos Teóricos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Espectrofotometría , Triptófano Sintasa/antagonistas & inhibidores , Triptófano Sintasa/genética , beta-Alanina/análogos & derivados , beta-Alanina/farmacologíaRESUMEN
Monoclonal antibodies directed against the native form of the beta 2 subunit of Escherichia coli tryptophan synthase strongly inhibit both its tryptophan synthase and its serine deaminase activities. The mechanism of this inactivation is studied here, by monitoring quantitatively the absorption and fluorescence properties of different well-characterized successive intermediates in the catalytic cycle of tryptophan synthase. It is shown that the antibodies interfere specifically with the formation of one or the other of these intermediates. It is concluded that the antibodies either modify or block the molecular flexibility of the protein, thus preventing conformational changes that the protein has to undergo during the catalysis. At least two different stages of the catalytic process, each one sensitive to a different class of antibodies, are shown to involve molecular movements of the polypeptide chain. Indications are given on the regions of the molecule involved in these movements.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Escherichia coli/enzimología , Triptófano Sintasa/antagonistas & inhibidores , Catálisis , Cromatografía Líquida de Alta Presión , Epítopos/inmunología , Fragmentos Fab de Inmunoglobulinas/farmacología , Inmunoglobulina G/farmacología , Indoles/metabolismo , L-Serina Deshidratasa/antagonistas & inhibidores , Sustancias Macromoleculares , Espectrometría de Fluorescencia , Triptófano Sintasa/inmunología , Triptófano Sintasa/metabolismoRESUMEN
Oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan, which are analogs of a proposed reaction intermediate, are potent competitive inhibitors of both tryptophanase and the alpha 2 beta 2 complex of tryptophan synthase (Phillips, R. S., Miles, E. W., and Cohen, L. A. (1984) Biochemistry 23, 6228-6234). Since these inhibitors can exist in two diastereoisomeric forms, which we expected to differ in inhibitory potency, we have separated the diastereoisomers of 2,3-dihydro-L-tryptophan by preparative high performance liquid chromatography. These diastereoisomers were designated "A" and "B" in order of elution from the high performance liquid chromatography column. Diastereoisomer B is a potent competitive inhibitor of the alpha 2 beta 2 complex of tryptophan synthase with KI = 6 microM at pH 7.8 and 25 degrees C. In contrast, diastereoisomer A is a weak competitive inhibitor, with KI = 940 microM under these conditions. With tryptophanase, the situation is reversed; diastereoisomer A is a potent slow-binding competitive inhibitor of tryptophanase with KI = 2 microM at pH 8.0 and 25 degrees C, while diastereoisomer B is much weaker with KI = 1600 microM under these conditions. These results not only provide additional support for the proposal that the indolenine tautomer of tryptophan is an intermediate in the reactions catalyzed by both enzymes but also suggest that these enzymes catalyze their respective reactions via enantiomeric indolenine intermediates.