RESUMEN
Vulnerable patients are at risk for neuroinflammation-mediated post-operative complications, including depression (POD) and cognitive dysfunction (POCD). Zucker rats, expressing multiple risk factors for post-operative complications in humans, may provide a clinically relevant model to study pathophysiology and explore potential interventions. J147, a newly developed anti-dementia drug, was shown to prevent POCD in young healthy rats, and improved early post-surgical recovery in Zucker rats. Aim of the present study was to investigate POCD and the therapeutic potential of J147 in male Zucker rats. Risk factors in the Zucker rat strain were evaluated by comparison with lean littermates. Zucker rats were subjected to major abdominal surgery. Acute J147 treatment was provided by a single iv injection (10 mg/kg) at the start of surgery, while chronic J147 treatment was provided in the food (aimed at 30 mg/kg/day), starting one week before surgery and up to end of protocol. Effects on behavior were assessed, and plasma, urine and brain tissue were collected and processed for immunohistochemistry and molecular analyses. Indeed, Zucker rats displayed increased risk factors for POCD, including obesity, high plasma triglycerides, low grade systemic inflammation, impaired spatial learning and decreased neurogenesis. Surgery in Zucker rats reduced exploration and increased anxiety in the Open Field test, impaired short-term spatial memory, induced a shift in circadian rhythm and increased plasma neutrophil gelatinase-associated lipocalin (NGAL), microglia activity in the CA1 and blood brain barrier leakage. Chronic, but not acute J147 treatment reduced anxiety in the Open Field test and protected against the spatial memory decline. Moreover, chronic J147 increased glucose sensitivity. Acute J147 treatment improved long-term spatial memory and reversed the circadian rhythm shift. No anti-inflammatory effects were seen for J147. Although Zucker rats displayed risk factors, surgery did not induce extensive POCD. However, increased anxiety may indicate POD. Treatment with J147 showed positive effects on behavioral and metabolic parameters, but did not affect (neuro)inflammation. The mixed effect of acute and chronic treatment may suggest a combination for optimal treatment.
Asunto(s)
Trastornos del Conocimiento , Humanos , Ratas , Masculino , Animales , Trastornos del Conocimiento/etiología , Ratas Zucker , Ratas Wistar , Cognición , Complicaciones Posoperatorias/etiología , Complicaciones Posoperatorias/psicología , Ansiedad/etiología , Inflamación/complicacionesRESUMEN
The crystal structures and enzymic properties of two mutant dihydrofolate reductases (Escherichia coli) were studied in order to clarify the functional role of an invariant carboxylic acid (aspartic acid at position 27) at the substrate binding site. One mutation, constructed by oligonucleotide-directed mutagenesis, replaces Asp27 with asparagine; the other is a primary-site revertant to Ser27. The only structural perturbations involve two internally bound water molecules. Both mutants have low but readily measurable activity, which increases rapidly with decreasing pH. The mutant enzymes were also characterized with respect to relative folate: dihydrofolate activities and kinetic deuterium isotope effects. It is concluded that Asp27 participates in protonation of the substrate but not in electrostatic stabilization of a positively charged, protonated transition state.
Asunto(s)
Ácido Aspártico , Tetrahidrofolato Deshidrogenasa , Cristalografía , Deuterio , Escherichia coli , Cinética , Metotrexato/metabolismo , Mutación , Unión Proteica , Relación Estructura-ActividadRESUMEN
Three mutations of the enzyme dihydrofolate reductase were constructed by oligonucleotide-directed mutagenesis of the cloned Escherichia coli gene. The mutations--at residue 27, aspartic acid replaced with asparagine; at residue 39, proline replaced with cysteine; and at residue 95, glycine replaced with alanine--were designed to answer questions about the relations between molecular structure and function that were raised by the x-ray crystal structures. Properties of the mutant proteins show that Asp-27 is important for catalysis and that perturbation of the local structure at a conserved cis peptide bond following Gly-95 abolishes activity. Substitution of cysteine for proline at residue 39 results in the appearance of new forms of the enzyme that correspond to various oxidation states of the cysteine. One of these forms probably represents a species cross-linked by an intrachain disulfide bridge between the cysteine at position 85 and the new cysteine at position 39.
Asunto(s)
Mutación , Tetrahidrofolato Deshidrogenasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Disulfuros , Escherichia coli/genética , Regulación de la Expresión Génica , Genes , Genes Bacterianos , Relación Estructura-ActividadRESUMEN
BACKGROUND: Angiogenesis is involved in tumor growth, macular degeneration, retinopathy and other diseases. Vascular endothelial growth factor (VEGF) stimulates angiogenesis by binding to specific receptors (VEGFRs) on the surface of vascular endothelial cells. VEGFRs are receptor tyrosine kinases that, like the platelet-derived growth factor receptors (PDGFRs), contain a large insert within the kinase domain. RESULTS: We report here the generation, kinetic characterization, and 2.4 A crystal structure of the catalytic kinase domain of VEGF receptor 2 (VEGFR2). This protein construct, which lacks 50 central residues of the 68-residue kinase insert domain (KID), has comparable kinase activity to constructs containing the entire KID. The crystal structure, determined in an unliganded phosphorylated state, reveals an overall fold and catalytic residue positions similar to those observed in other tyrosine-kinase structures. The kinase activation loop, autophosphorylated on Y1059 prior to crystallization, is mostly disordered; however, a portion of it occupies a position inhibitory to substrate binding. The ends of the KID form a beta-like structure, not observed in other known tyrosine kinase structures, that packs near to the kinase C terminus. CONCLUSIONS: The majority of the VEGFR2 KID residues are not necessary for kinase activity. The unique structure observed for the ends of the KID may also occur in other PDGFR family members and may serve to properly orient the KID for signal transduction. This VEGFR2 kinase structure provides a target for design of selective anti-angiogenic therapeutic agents.
Asunto(s)
Neovascularización Fisiológica , Conformación Proteica , Proteínas Tirosina Quinasas Receptoras/química , Receptores de Factores de Crecimiento/química , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Sustancias de Crecimiento/química , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Familia de Multigenes , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento/fisiología , Receptores de Factores de Crecimiento Endotelial Vascular , Proteínas Recombinantes de Fusión/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The structure of the Escherichia coli thymidylate synthase (TS) covalent inhibitory ternary complex consisting of enzyme, 5-fluoro-2'-deoxyuridylate (FdUMP) and 5,10-methylene tetrahydrofolate (CH2-H4PteGlu) has been determined at 2.5 A resolution using difference Fourier methods. This complex is believed to be a stable structural analog of a true catalytic intermediate. Knowledge of its three-dimensional structure and that for the apo enzyme, also reported here, suggests for the first time how TS may activate dUMP and CH2-H4PteGlu leading to formation of the intermediate and offers additional support for the hypothesis that the substrate and cofactor are linked by a methylene bridge between C-5 of the substrate nucleotide and N-5 of the cofactor. By correlating these structural results with the known stereospecificity of the TS-catalyzed reaction it can be inferred that the catalytic intermediate, once formed, must undergo a conformational isomerization before eliminating across the bond linking C-5 of dUMP to C-11 of the cofactor. The elimination itself may be catalyzed by proton transfer to the cofactor's 5 nitrogen from invariant Asp169 buried deep in the TS active site. The juxtaposition of Asp169 and bound tetrahydrofolate in TS is remarkably reminiscent of binding geometry found in dihydrofolate reductase where a similarly conserved carboxyl group serves as a general acid for protonating the corresponding pyrazine ring nitrogen of dihydrofolate.
Asunto(s)
Nucleótidos de Desoxiuracil/metabolismo , Fluorodesoxiuridilato/metabolismo , Antagonistas del Ácido Fólico/metabolismo , Tetrahidrofolatos/metabolismo , Timidilato Sintasa/metabolismo , Sitios de Unión , Escherichia coli/enzimología , Fluorodesoxiuridilato/farmacología , Enlace de Hidrógeno , Ligandos , Modelos Moleculares , Conformación Proteica , Tetrahidrofolatos/farmacología , Timidilato Sintasa/antagonistas & inhibidores , Difracción de Rayos X/métodosRESUMEN
In the past year, the three-dimensional structures of two serine/threonine phosphatases, protein phosphatase-1 and protein phosphatase-2b (calcineurin), have been determined. The new information puts previous sequence comparisons and mutagenesis studies into a detailed structural perspective. The active-site structure and catalytic mechanism appear to be common to a variety of phosphoesterase enzymes.
Asunto(s)
Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/fisiología , Secuencia de Aminoácidos , Calcineurina , Proteínas de Unión a Calmodulina/química , Proteínas de Unión a Calmodulina/genética , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/clasificación , Fosfoproteínas Fosfatasas/genética , Conformación Proteica , Ingeniería de Proteínas , Proteína Fosfatasa 1 , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Serina/metabolismo , Treonina/metabolismoRESUMEN
To develop novel lipophilic thymidylate synthase (TS) inhibitors, the X-ray structure of Escherichia coli TS in ternary complex with FdUMP and the inhibitor 10-propargyl-5,8-dideazafolic acid (CB3717) was used as a basis for structure-based design. A total of 31 novel lipophilic TS inhibitors, lacking a glutamate residue, were synthesized; 26 of them had in common a N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2-ynylaniline+ ++ structure in which the aniline was appropriately substituted with simple lipophilic substituents either in position 3 or 4, or in both. Compounds were tested for their inhibition of E. coli TS and human TS and also for their inhibition of the growth in tissue culture of a murine leukemia, a human leukemia, and a thymidine kinase-deficient human adenocarcinoma. The crystal structures of five inhibitors complexed with E. coli TS were determined. Five main conclusions are drawn from this study. (i) A 3-substituent such as CF(3), iodo, or ethynyl enhances binding by up to 1 order of magnitude and in the case of CF(3) was proven to fill a nearby pocket in the enzyme. (ii) A simple strongly electron-withdrawing substituent such as NO(2) or CF(3)SO(2) in the 4-position enhances binding by 2 orders of magnitude; it is hypothesized that the transannular dipole so induced interacts favorably with the protein. (iii) Attempts to combine the enhancements of i and ii in the same molecule were generally unsuccessful (iv) A 4-C(6)H(5)SO(2) substituent provided both electron withdrawal and a van der Waal's interaction of the phenyl group with a hydrophobic surface at the mouth of the active site. The inhibition (K(is) = 12 nM) of human TS by this compound, 7n, showed that C(6)H(5)SO(2) provided virtually as much binding affinity as the CO-glutamate which it had replaced. (v) The series of compounds were poorly water soluble, and also the potent TS inhibition shown by several of them did not translate into good cytotoxicity. Compounds with large cyclic groups linked to position 4 by an SO or SO(2) group did, however, have IC(50)'s in the range 1-5 microM. Of these, 4-(N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2-ynylamino )phenyl phenyl sulfone, 7n, had IC(50)'s of about 1 microM and was chosen for further elaboration.
Asunto(s)
Antineoplásicos/síntesis química , Inhibidores Enzimáticos/síntesis química , Antagonistas del Ácido Fólico/síntesis química , Quinazolinas/síntesis química , Timidilato Sintasa/antagonistas & inhibidores , Adenocarcinoma , Animales , Antineoplásicos/química , Antineoplásicos/toxicidad , Sitios de Unión , Línea Celular , Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Escherichia coli/enzimología , Antagonistas del Ácido Fólico/química , Antagonistas del Ácido Fólico/farmacología , Humanos , Leucemia , Leucemia L1210 , Ratones , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Conformación Proteica , Quinazolinas/química , Quinazolinas/farmacología , Relación Estructura-Actividad , Timidilato Sintasa/química , Células Tumorales CultivadasRESUMEN
The structure-based design and subsequent chemical synthesis of novel, urea-containing FKBP12 inhibitors are described. These compounds are shown to disrupt the cis-trans peptidylprolyl isomerase activity of FKBP12 with inhibition constants (Ki,app) approaching 0.10 microM. Analyses of several X-ray crystal structures of FKBP12-urea complexes demonstrate that the urea-containing inhibitors associate with FKBP12 in a manner that is similar to, but significantly different from, that observed for the natural product FK506.
Asunto(s)
Proteínas Portadoras/antagonistas & inhibidores , Proteínas de Unión al ADN/antagonistas & inhibidores , Diseño de Fármacos , Proteínas de Choque Térmico/antagonistas & inhibidores , Urea/análisis , Isomerasas de Aminoácido/antagonistas & inhibidores , Secuencia de Aminoácidos , Proteínas Portadoras/química , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Proteínas de Choque Térmico/química , Humanos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Isomerasa de Peptidilprolil , Relación Estructura-Actividad , Tacrolimus/química , Proteínas de Unión a TacrolimusAsunto(s)
Antagonistas del Ácido Fólico/metabolismo , Ácido Fólico/análogos & derivados , Timidilato Sintasa/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cisteína , Escherichia coli/enzimología , Escherichia coli/genética , Ácido Fólico/metabolismo , Ácido Fólico/farmacología , Antagonistas del Ácido Fólico/farmacología , Cinética , Mutagénesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Timidilato Sintasa/química , Timidilato Sintasa/aislamiento & purificaciónRESUMEN
The cytotoxin ricin is a heterodimer composed of an A chain which enzymatically inhibits protein synthesis on eukaryotic ribosomes and a lectin B chain which binds to cell surfaces and triggers uptake of the toxin. A low resolution (4 A) x-ray structure revealed that the B chain is a bilobal structure and that each domain binds a galactose sugar. These apparent structural similarities suggested that the protein might have internal symmetry and might have arisen by gene duplication. A subsequent search of the amino acid sequence provided strong evidence for homology between th NH2- and COOH-terminal halves of the B chain and suggested that they may have arisen from a common ancestor. There is no strong relationship between the halves of the A chain and little, if any, significant sequence homology between the A and B chains of ricin.
Asunto(s)
Ricina , Secuencia de Aminoácidos , Galactosa , Sustancias Macromoleculares , Modelos Moleculares , Unión Proteica , Conformación Proteica , Ricina/genética , Difracción de Rayos XRESUMEN
We have determined kinetic and thermodynamic constants governing binding of substrates and products to thymidylate synthase from Escherichia coli (TS) sufficient to describe the kinetic scheme for this enzyme. (1) The catalytic mechanism is ordered in the following manner, TS + dUMP --> TS x dUMP + (6R)-5,10-CH2-H4folate --> TS x dUMP x (6R)-5,10-CH2H4folate --> TS x dTMP x H2folate --> TS x dTMP --> TS as predicted previously by others from steady-state measurements. (2) When substrates are saturating, the overall reaction rate is governed by the slow conversion of enzyme-bound substrates to bound products as demonstrated by (i) large primary and secondary isotope effects on k(cat) and (ii) high rates of product dissociation compared to k(cat). (3) Stopped-flow studies measuring the binding of 10-propargyl-5,8-dideazafolate, an analog of (6R)-5,10-CH2H4folate, with the active site mutant C146A or the C-terminus-truncated mutant P261Am enabled us to identify physical events corresponding to spectral changes which are observed with the wild-type enzyme during initiation of catalysis. A kinetically identifiable reaction step, TS x dUMP x (6R)-5,10-CH2H4folate --> (TS x dUMP x (6R)-5,10-CH2H4folate)*, likely represents reorientation of the C-terminus of the enzyme over the catalytic site. This seals the substrates into a relatively nonaqueous environment in which catalysis can occur. (4) Although TS is a dimer of identical subunits, catalysis is probably confined to only one subunit at a time. (5) The "high-resolution" kinetic scheme described herein provides a framework for the interpretation of the kinetics of catalysis by mutant ecTS chosen to provide insights into the relationship between structure and function.
Asunto(s)
Escherichia coli/enzimología , Ácido Fólico/metabolismo , Tetrahidrofolatos/metabolismo , Timidilato Sintasa/metabolismo , Catálisis , Nucleótidos de Desoxiuracil/metabolismo , Ácido Fólico/análogos & derivados , Ácido Fólico/farmacología , Antagonistas del Ácido Fólico/farmacología , Cinética , Ligandos , Estructura Molecular , Mutagénesis Sitio-Dirigida/genética , Unión Proteica , Conformación Proteica , Quinazolinas/farmacología , Espectrometría de Fluorescencia , Espectrofotometría , Termodinámica , Timidina Monofosfato/metabolismo , Timidilato Sintasa/químicaRESUMEN
The recent determination of the crystal structure of Escherichia coli thymidylate synthase (TS) [Matthews et al. (1989) J. Mol. Biol. 205, 449-454] has implicated the glutamic acid residue at position 58 in a mechanistic role which could involve the interaction of its gamma-carboxyl side chain with the nucleotide substrate and/or the folate cofactor. The site-specific mutagenesis of Glu-58 to Gln-58 in E. coli TS provided the opportunity to explore its functional role in activity and binding. When profiled by the spectrophotometric and tritium release assays, the 370- and 760-fold decreases, respectively, in kcat and the elevated Km values for the Gln-58 mutant enzyme indicated a significant involvement of Glu-58 in substrate binding and turnover. The apparent dissociation constant for the covalent FdUMP-enzyme binary complex was 30 microM, which is 5-fold higher than that found for the wild-type enzyme, while the inhibitory ternary complex apparent dissociation constants for FdUMP and CH2H4folate for the Gln-58 enzyme were 10- and 60-fold higher, respectively, than those for the wild-type enzyme under saturating conditions. The extent of covalent FdUMP binding to the Gln-58 enzyme was reduced from 1.5 to 0.7 per dimer in the inhibitory ternary complex but only from 0.7 to 0.5 per dimer in the binary complex of the Gln-58 enzyme. The usual 2.1-fold enhancement of FdUMP binding to wild-type TS in the presence of CH2H4folate was not observed for the Gln-58 enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Escherichia coli/enzimología , Glutamatos/metabolismo , Glutamina/metabolismo , Timidilato Sintasa/metabolismo , Sitios de Unión , Catálisis , Ácido Glutámico , Cinética , Ligandos , Espectroscopía de Resonancia Magnética , Mutagénesis Sitio-Dirigida , Timidilato Sintasa/genética , Timidilato Sintasa/aislamiento & purificaciónRESUMEN
Using oligonucleotide-directed site-specific mutagenesis, we have constructed a system for the mutation and expression of yeast cytochrome c peroxidase (CCP, EC 1.11.1.5) in Escherichia coli and applied it to test the hypothesis that Trp-51 is the locus of the free radical observed in compound I of CCP [Poulos, T. L., & Kraut, J. (1980) J. Biol. Chem. 255, 8199-8205]. The system was created by substituting a CCP gene modified by site-directed mutagenesis, CCP(MI), for the fol gene in a vector previously used for mutagenesis and overexpression of dihydrofolate reductase. E. coli transformed with the resulting plasmid produced the CCP(MI) enzyme in large quantities, more than 15 mg/L of cell culture, of which 10% is holo- and 90% is apo-CCP(MI). The apoenzyme was easily converted to holoenzyme by the addition of bovine hemin. Purified CCP(MI) has the same catalytic activity and spectra as bakers' yeast CCP. A mutation has been made in CCP(MI), Trp-51 to Phe. The Phe-51 mutant protein CCP(MI,F51) is fully active, and the electron paramagnetic resonance (EPR) spectrum, at 89 K, of its oxidized intermediate, compound I, displays a strong sharp resonance at g = 2.004, which is very similar to the signal observed for compound I of both bakers' yeast CCP and CCP(MI). However, UV-visible and EPR spectroscopy revealed that the half-life of CCP(MI,F51) compound I at 23 degrees C is only 1.4% of that observed for the compound I forms of CCP(MI) or bakers' yeast CCP. Thus, Trp-51 is not necessary for the formation of the free radical observed in compound I but appears to exert a significant influence on its stability.
Asunto(s)
Citocromo-c Peroxidasa/genética , Escherichia coli/genética , Mutación , Peroxidasas/genética , Saccharomyces cerevisiae/genética , Transcripción Genética , Triptófano , Secuencia de Aminoácidos , Secuencia de Bases , Enzimas de Restricción del ADN , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres , Plásmidos , Saccharomyces cerevisiae/enzimologíaRESUMEN
Substitution of cysteine for proline-39 in Escherichia coli dihydrofolate reductase by oligonucleotide-directed mutagenesis positions the new cysteine adjacent to already existing cysteine-85. When the mutant protein is expressed in the E. coli cytosol, the cysteine sulfur atoms are found, by X-ray crystallographic analysis, to be in van der Waals contact but not covalently bonded to one another. In vitro oxidation by dithionitrobenzoate results in formation of a disulfide bond between residues 39 and 85 with a geometry close to that of the commonly observed left-handed spiral. Comparison of 2.0-A-refined crystal structures of the oxidized (cross-linked) and reduced (un-cross-linked) forms of the mutant enzyme shows that the conformation of the enzyme molecule was not appreciably affected by formation of the disulfide bond but that details of the molecule's thermal motion were altered. The disulfide-cross-linked enzyme is at least 1.8 kcal/mol more stable with respect to unfolding, as measured by guanidine hydrochloride denaturation, than either the wild-type or the reduced (un-cross-linked) mutant enzyme. Nevertheless, the cross-linked form is not more resistant to thermal denaturation. Moreover, the appearance of intermediates in the guanidine hydrochloride denaturation profile and urea-gradient polyacrylamide gels indicates that the folding/unfolding pathway of the disulfide-cross-linked enzyme has changed significantly.
Asunto(s)
Disulfuros/análisis , Escherichia coli/genética , Genes Bacterianos , Genes , Mutación , Tetrahidrofolato Deshidrogenasa/genética , Secuencia de Aminoácidos , Clonación Molecular , Cisteína , Escherichia coli/enzimología , Guanidina , Guanidinas/farmacología , Modelos Moleculares , Conformación Proteica , Tetrahidrofolato Deshidrogenasa/metabolismo , Difracción de Rayos XRESUMEN
We have explored the substrate protonation mechanism of Escherichia coli dihydrofolate reductase by changing the location of the proton donor. A double mutant was constructed in which the proton donor of the wild-type enzyme, aspartic acid-27, has been changed to serine and simultaneously an alternative proton donor, glutamic acid, has replaced threonine at position 113. The active site of the resulting variant enzyme molecule should therefore somewhat resemble that proposed for the R67 plasmid-encoded dihydrofolate reductase [Matthews, D. A., Smith, S. L., Baccanari, D. P., Burchall, J. J., Oatley, S. J., & Kraut, J. (1986) Biochemistry 25, 4194]. At pH 7, the double-mutant enzyme has a 3-fold greater kcat and an unchanged Km(dihydrofolate) as compared with the single-mutant Asp-27----Ser enzyme described previously [Howell, E. E., Villafranca, J. E., Warren, M. S., Oatley, S. J., & Kraut, J. (1986) Science (Washington, D.C.) 231, 1123]. Additionally, its activity vs pH profiles together with observed deuterium isotope effects, suggest that catalysis depends on an acidic group with a pKa of 8. It is concluded that the dihydropteridine ring of a bound substrate molecule can indeed be protonated by a glutamic acid side chain at position 113 (instead of an aspartic acid side chain at position 27), but with greatly decreased efficiency: at pH 7, the double mutant still has a 25-fold lower kcat (1.2 s-1) and a 2900-fold lower kcat/km(dihydrofolate) (8.6 X 10(3) s-1 M-1) than the wild-type enzyme.
Asunto(s)
Escherichia coli/enzimología , Tetrahidrofolato Deshidrogenasa/metabolismo , Escherichia coli/genética , Genes , Genes Bacterianos , Glutamatos , Ácido Glutámico , Concentración de Iones de Hidrógeno , Cinética , Matemática , Metotrexato/metabolismo , Mutación , Unión Proteica , Protones , Serina , Tetrahidrofolato Deshidrogenasa/genéticaRESUMEN
The steadily increasing number of high-resolution human immunodeficiency virus (HIV) 1 protease complexes has been the impetus for the elaboration of knowledge-based mean field ligand-protein interaction potentials. These potentials have been linked with the hydrophobicity and conformational entropy scales developed originally to explain protein folding and stability. Empirical free energy calculations of a diverse set of HIV-1 protease crystallographic complexes have enabled a detailed analysis of binding thermodynamics. The thermodynamic consequences of conformational changes that HIV-1 protease undergoes upon binding to all inhibitors, and a substantial concomitant loss of conformational entropy by the part of HIV-1 protease that forms the ligand-protein interface, have been examined. The quantitative breakdown of the entropy-driven changes occurring during ligand-protein association, such as the hydrophobic contribution, the conformational entropy term and the entropy loss due to a reduction of rotational and translational degrees of freedom, of a system composed to ligand, protein and crystallographic water molecules at the ligand-protein interface has been carried out. The proposed approach provides reasonable estimates of distinctions in binding affinity and gives an insight into the nature of enthalpyentropy compensation factors detected in the binding process.
Asunto(s)
Proteasa del VIH/metabolismo , Cristalización , Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/química , Humanos , Ligandos , Modelos Químicos , Oligopéptidos/química , Unión Proteica , Especificidad por Sustrato , Termodinámica , Agua/químicaRESUMEN
Resonance Raman (RR) spectra are reported for the ternary complex of Escherichia coli thymidylate synthase with the cofactor 5,10-methylenetetrahydrofolate (CH2-H4-folate) and the inhibitor 5-fluoro-2'-deoxyuridylate, excited at 337 or 356 nm, in resonance with perturbed absorption bands of the p-aminobenzoylglutamate (PABA-Glu) portion of the cofactor. For comparison, RR spectra were obtained with 260 nm excitation for PABA-Glu in various solvents, and for CH2H4-folate and H4-folate in aqueous solution. These reference spectra are assigned to modes of PABA-Glu in its benzenoid form. The ternary complex RR spectra are very different, however, and are assigned, with the aid of isotopic data, to the PABA-Glu in a predominantly quinoid form. Similar spectra were obtained for the ternary complexes of the E58Q and K48Q mutants, indicating that neither Glu58 nor Lys48 are essential for maintaining the quinoid structure, even though their side chains complement the dipolar charge distribution of the quinoid form of PABA-Glu. Since these are the only charged residues in the PABA-Glu vicinity, electrostatic stabilization is not essential to maintenance of the quinoid structure. It is proposed that quinoid formation results from steric forces, probably resulting from the protein conformation change known to accompany cofactor binding, which enforce coplanarity of the PABA-Glu ring and substituents. This stereoelectronic change activates the cofactor by opening the methylene bridge. A second RR spectrum of the ternary complex, previously proposed to reflect an alternate structure, is shown to result instead from irreversible formation of a laser-induced photoproduct.
Asunto(s)
Tetrahidrofolatos/química , Timidilato Sintasa/metabolismo , Escherichia coli/enzimología , Espectrometría RamanRESUMEN
Resonance Raman (RR) spectra are reported for the binary complex of Escherichia coli thymidylate synthase (TS) with the substrate analog inhibitor 5-nitrodeoxyuridylate (NDU). The TS/NDU binary complex RR spectrum shows many similarities to the RR spectra of thiol adducts of NDU or of 5-nitro-1-methyluracil formed in solution, providing strong evidence in support of the formation of a covalent link between Cys146 of TS and C6 of NDU. Spectral differences between the model compounds and the binary complex reflect the consequences of fixing the conformations of the uracil and ribose rings at the enzyme active site. The RR spectra of the ternary complexes of TS/NDU with either tetrahydrofolate (H4-folate) or the cofactor 5,10-methylenetetrahydrofolate (CH2H4-folate) show that a covalent link is not formed between C11 of CH2H4-folate and C5 of NDU. Neither does the methylene bridge of CH2H4-folate remain intact in the ternary complex; either CH2H4-folate is present as the N5 iminium cation species or the methylene group is lost as formaldehyde. A shift in the NO2 symmetric stretching frequency in the ternary complex indicates expulsion of water molecules from the region of the NO2 group by the cofactor.
Asunto(s)
Nucleótidos de Desoxiuracil/química , Timidilato Sintasa/química , Escherichia coli/enzimología , Espectrofotometría Ultravioleta , Espectrometría RamanRESUMEN
Replacement of methionine (Met) residues by selenomethionine (SeMet) was recently shown to facilitate the crystallographic analysis of protein structure through the application of multi-wavelength anomalous diffraction techniques [Yang et al. (1990) Science (Washington, D.C.) 249, 1398-1405]. The availability of SeMet-containing proteins provides an excellent opportunity to evaluate the effects of the complete replacement of Met by SeMet. We chose to compare the properties of selenomethionyl thymidylate synthase isolated from Escherichia coli DL41 (a methionine auxotroph) and wild-type (wt) enzyme obtained from E. coli Rue10. An improved purification procedure for thymidylate synthase was developed which permitted the isolation of 25 mg of pure protein from 2 g of E. coli in 90% yield in no more than 8 h. The pure wt and SeMet enzymes exhibited specific activities 40% higher than published values. Thermal stability studies at 30 degrees C in degassed buffer showed that the SeMet enzyme (t1/2 67 h) was 8-fold less stable than wt enzyme (t1/2 557 h). The half-lives for the latter enzymes in nondegassed buffers at 30 degrees C were decreased by 2-fold, thus indicating the sensitivity of the enzyme to dissolved oxygen. Both enzymes exhibited essentially the same kinetic and binding properties, including Km(dUMP) (1.2 x 10(-6) M), specificity constant (1.6 x 10(6) s-1 M-1), and Kd for 5-fluorodeoxyuridylate binding (1.2 nM) in covalent inhibitory ternary complexes. In addition, X-ray crystallographic analysis by difference Fourier synthesis showed there was no significant difference in conformation between the SeMet enzyme and the wt enzyme.