RESUMEN
N9-GP/Rebinyn®/Refixia® is an approved PEGylated (polyethylene glycol-conjugated) recombinant human factor IX intended for prophylactic and/or on-demand treatment in adults and children with haemophilia B. A juvenile neurotoxicity study was conducted in male rats to evaluate effects on neurodevelopment, sexual maturation, and fertility following repeat-dosing of N9-GP. Male rats were dosed twice weekly from Day 21 of age with N9-GP or vehicle for 10 weeks, followed by a dosing-free recovery period for 13 weeks and terminated throughout the dosing and recovery periods. Overall, dosing N9-GP to juvenile rats did not result in any functional or pathological effects, as measured by neurobehavioural/neurocognitive tests, including motor activity, sensory function, learning and memory as well as growth, sexual maturation, and fertility. This was further supported by the extensive histopathologic evaluation of brain tissue. Exposure and distribution of polyethylene glycol was investigated in plasma, choroid plexus, cerebrospinal fluid, and brain sections. PEG did not cross the blood brain barrier and PEG exposure did not result in any effects on neurodevelopment. In conclusion, dosing of N9-GP to juvenile rats did not identify any effects on growth, sexual maturation and fertility, clinical and histological pathology, or neurodevelopment related to PEG exposure and supports the prophylactic use of N9-GP in children.
Asunto(s)
Factor IX , Hemofilia B , Adulto , Animales , Niño , Factor IX/uso terapéutico , Fertilidad , Hemofilia B/tratamiento farmacológico , Humanos , Lactante , Masculino , Polietilenglicoles/toxicidad , Ratas , Proteínas RecombinantesRESUMEN
Oligonucleotides as short as 6 nt in length have been shown to bind specifically and tightly to proteins and affect their biological function. Yet, sparse structural data are available for corresponding complexes. Employing a recently developed hexanucleotide array, we identified hexadeoxyribonucleotides that bind specifically to the 3C protease of hepatitis A virus (HAV 3C(pro)). Inhibition assays in vitro identified the hexanucleotide 5'-GGGGGT-3' (G(5)T) as a 3C(pro) protease inhibitor. Using (1)H NMR spectroscopy, G(5)T was found to form a G-quadruplex, which might be considered as a minimal aptamer. With the help of (1)H, (15)N-HSQC experiments the binding site for G(5)T was located to the C-terminal ß-barrel of HAV 3C(pro). Importantly, the highly conserved KFRDI motif, which has previously been identified as putative viral RNA binding site, is not part of the G(5)T-binding site, nor does G(5)T interfere with the binding of viral RNA. Our findings demonstrate that sequence-specific nucleic acid-protein interactions occur with oligonucleotides as small as hexanucleotides and suggest that these compounds may be of pharmaceutical relevance.
Asunto(s)
Cisteína Endopeptidasas/química , Desoxirribonucleótidos/química , Virus de la Hepatitis A/enzimología , Proteínas Virales/química , Proteasas Virales 3C , Antivirales/química , Sitios de Unión , Cisteína Endopeptidasas/metabolismo , Desoxirribonucleótidos/metabolismo , Dimerización , G-Cuádruplex , Resonancia Magnética Nuclear Biomolecular , ARN Viral/metabolismo , Proteínas Virales/metabolismoRESUMEN
NMR artifact purging: Modern NMR experiments depend on efficient coherence transfer pathways for their sensitivity and on suppression of undesired pathways leading to artifacts for their spectral clarity. A novel robust adiabatic element suppresses hard-to-get-at artifacts (see picture).
Asunto(s)
Artefactos , Resonancia Magnética Nuclear Biomolecular/métodos , Isótopos de Carbono/química , Modelos Químicos , Rafinosa/química , Procesamiento de Señales Asistido por ComputadorRESUMEN
Saturation transfer difference (STD) NMR experiments on human N-acetylglucosamine kinase (GlcNAc kinase) have been used to determine binding epitopes for the GlcNAc and ATP substrates and their analogues. The study reveals that during the enzyme reaction the binding mode of both substrates is conserved, although the binding affinity of the sugar is reduced. This suggests that the protein does not undergo any significant structural changes during catalysis. Our experiments also demonstrate that GlcNAc kinase has residual activity in the absence of Mg(2+). Furthermore, our experiments clearly show that the GlcNAc kinase predominately, if not exclusively, produces the beta anomer of phosphorylated sugars. To identify the minimum requirements for substrate binding, a detailed analysis of different natural occurring as well as synthetic sugars was employed. Modifications at the 1, 2, 3, 4 and 6 position as well as the N-acetyl group greatly reduce the binding affinity. In addition, the binding mode of these substrate analogues is often also changed. The high beta anomeric preference of GlcNAc kinase along with the drastically reduced binding affinity for sugars other than GlcNAc, suggests that GlcNAc kinase phosphorylates beta-GlcNAc in cells.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Adenosina Difosfato/química , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Sitios de Unión , Dimerización , Humanos , Ligandos , Magnesio/química , Estructura Molecular , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Soluciones/química , Especificidad por Sustrato , Agua/químicaRESUMEN
Oral administration of therapeutic peptides is hindered by poor absorption across the gastrointestinal barrier and extensive degradation by proteolytic enzymes. Here, we investigated the absorption of orally delivered semaglutide, a glucagon-like peptide-1 analog, coformulated with the absorption enhancer sodium N-[8-(2-hydroxybenzoyl) aminocaprylate] (SNAC) in a tablet. In contrast to intestinal absorption usually seen with small molecules, clinical and preclinical dog studies revealed that absorption of semaglutide takes place in the stomach, is confined to an area in close proximity to the tablet surface, and requires coformulation with SNAC. SNAC protects against enzymatic degradation via local buffering actions and only transiently enhances absorption. The mechanism of absorption is shown to be compound specific, transcellular, and without any evidence of effect on tight junctions. These data have implications for understanding how highly efficacious and specific therapeutic peptides could be transformed from injectable to tablet-based oral therapies.
Asunto(s)
Receptor del Péptido 1 Similar al Glucagón/agonistas , Péptidos Similares al Glucagón/farmacología , Absorción Intestinal , Estómago/fisiología , Administración Oral , Adolescente , Adulto , Anciano , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Perros , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/ultraestructura , Péptido 1 Similar al Glucagón/análogos & derivados , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Péptidos Similares al Glucagón/administración & dosificación , Péptidos Similares al Glucagón/farmacocinética , Humanos , Concentración de Iones de Hidrógeno , Masculino , Persona de Mediana Edad , Ratas , Estómago/efectos de los fármacos , Factores de Tiempo , Adulto JovenRESUMEN
A new 2D NMR experiment, HAT HMBC, that is a hybrid of H2BC and HMBC aims at establishing two-bond correlations absent in H2BC spectra because of vanishing (3)J(HH) coupling constants. The basic idea is to create an additional pi phase difference in the multiplet structure in HMBC peaks with respect to the (n+1)J(HH) coupling constant between the proton(s) attached to a (13)C and a (1)H separated by n bonds. Thus HMBC peaks associated with small J(HH) will be the most attenuated in a HAT HMBC spectrum in comparison to a regular HMBC spectrum, i.e. peaks associated with (n+1)J(HH) and (n)J(CH) will for n>2 usually be strongly attenuated. The HAT HMBC pulse sequences contain the same number of pulses as regular HMBC and are only a few milliseconds longer.
Asunto(s)
Algoritmos , Modelos Químicos , Resonancia Magnética Nuclear Biomolecular/métodos , Procesamiento de Señales Asistido por Computador , Simulación por Computador , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Carbohydrate-protein interactions are frequently characterized by dissociation constants in the microM to mM range. This is normally associated with fast dissociation rates of the corresponding complexes, in turn leading to fast exchange on the nuclear magnetic resonance (NMR) chemical shift time scale and on the NMR relaxation time scale. Therefore, NMR experiments that take advantage of fast exchange are well suited to study carbohydrate-protein interactions. In general, it is possible to analyze ligand binding by observing either protein signals or ligand resonances. Because most receptor proteins to which carbohydrates bind are rather large with molecular weights significantly exceeding 30 kDa, the analysis of the corresponding protein spectra is not trivial, and only very few studies have been addressing this issue so far. We, therefore, focus on NMR experiments that employ observation of free ligand, that is, carbohydrate signals to analyze the bound state. Two types of NMR experiments have been extremely valuable to analyze carbohydrate-protein interactions at atomic resolution. Whereas transferred nuclear Overhauser effect (NOE) experiments deliver bioactive conformations of carbohydrates binding to proteins, saturation transfer difference (STD) NMR spectra provide binding epitopes and valuable information about the binding thermodynamics and kinetics. We demonstrate the power of a combined transfer NOE/STD NMR approach for the analysis of carbohydrate-protein complexes using selected examples.
Asunto(s)
Conformación de Carbohidratos , Carbohidratos/química , Espectroscopía de Resonancia Magnética/métodos , Proteínas/química , Ligandos , Unión Proteica , Proteínas/metabolismoRESUMEN
Two novel pulse sequences, CALIS-1 and CALIS-2, for accurate calibration of the RF field strength for an indirectly observed spin are introduced. CALIS-2 is intended for calibration of e.g., (13)C or (15)N pulses on natural abundance samples whilst CALIS-1 is recommended primarily for enriched samples. Both experiments can be performed without prior knowledge or guess of the RF field strength and no delays in the pulse sequences are critically dependent on coupling constants.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/instrumentación , Péptidos/química , Calibración , Isótopos de Carbono , Manosa/química , Isótopos de Nitrógeno , Proteínas de Plantas , Ondas de RadioRESUMEN
A new set of pulse sequences, 2CALIS, that exhibit double sensitivity of the recent CALIS pulse sequences for accurate calibration of the rf field strength for an indirectly observed spin is introduced. The sensitivity gain is a result of not forming heteronuclear coherence transfer gradient echoes although they are excellent for artifact suppression. It is, however, demonstrated that the scheme in 2CALIS for suppression of non (13)C-attached proton magnetization is adequate for calibration of the (13)C rf field strength even on natural abundance samples. A 2CALIS version with Watergate applicable to biomolecules in aqueous solution is also presented and demonstrated both in (13)C natural abundance and on a (13)C, (15)N enriched protein sample.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Oligopéptidos/química , Artefactos , Calibración , Isótopos de Carbono , Radioisótopos de Carbono , Isótopos de Nitrógeno , Sensibilidad y EspecificidadRESUMEN
Proteinase 3C of hepatitis A virus (HAV) plays a key role in the viral life cycle by generating mature viral proteins from the precursor polyprotein. In addition to its proteolytic activity, 3C binds to viral RNA, and thus influences viral genome replication. In order to investigate the interplay between proteolytic activity and RNA binding at the molecular level, we subjected HAV 3C and three variants carrying mutations of the cysteine residues [C24S (Cys-24-->Ser), C172A and C24S/C172A] to proteolysis assays with peptide substrates, and to surface plasmon resonance binding studies with peptides and viral RNA. We report that the enzyme readily forms dimers via disulphide bridges involving Cys-24. Dissociation constants (K(D)) for peptides were in the millimolar range. The binding kinetics for the peptides were characterized by k(on) and k(off) values of the order of 10(2) M(-1) x s(-1) and 10(-2) to 10(-1) s(-1) respectively. In contrast, 3C binding to immobilized viral RNA, representing the structure of the 5'-terminal domain, followed fast binding kinetics with k(on) and k(off) values beyond the limits of the kinetic resolution of the technique. The affinity of viral RNA depended strongly on the dimerization status of 3C. Whereas monomeric 3C bound to the viral RNA with a K(D) in the millimolar range, dimeric 3C had a significantly increased binding affinity with K(D) values in the micromolar range. A model of the 3C dimer suggests that spatial proximity of the presumed RNA-binding motifs KFRDI is possible. 3C binding to RNA was also promoted in the presence of substrate peptides, indicating co-operativity between RNA binding and protease activity. The data imply that the dual functions of 3C are mutually dependent, and regulate protein and RNA synthesis during the viral life cycle.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Virus de la Hepatitis A/enzimología , ARN Viral/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Línea Celular Tumoral , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Dimerización , Enzimas Inmovilizadas , Virus de la Hepatitis A/genética , Humanos , Hidrólisis , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Modelos Moleculares , Mutación/genética , Resonancia Magnética Nuclear Biomolecular/métodos , Fragmentos de Péptidos/metabolismo , Unión Proteica/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidad por Sustrato/genéticaRESUMEN
A novel method, Spin-State-Selective (S(3)) HMBC, for accurate measurement of homonuclear coupling constants is introduced. As characteristic for S(3) techniques, S(3) HMBC yields independent subspectra corresponding to particular passive spin states and thus allows determination of coupling constants between detected spins and homonuclear coupling partners along with relative signs. In the presented S(3) HMBC experiment, spin-state selection occurs via large one-bond coupling constants ensuring high editing accuracy and unequivocal sign determination of the homonuclear long-range relative to the associated one-bond coupling constant. The sensitivity of the new experiment is comparable to that of regular edited HMBC and the accuracy of the J/RDC measurement is as usual for E.COSY and S(3)-type experiments independent of the size of the homonuclear coupling constant of interest. The merits of the method are demonstrated by an application to strychnine where thirteen J(HH) coupling constants not previously reported could be measured.
RESUMEN
A specific covalently linked dimeric species of insulin high molecular weight products (HMWPs), formed during prolonged incubation of a neutral pharmaceutical formulation of human insulin, were characterized in terms of tertiary structure, self-association, biological activity, and fibrillation properties. The dimer was formed by a covalent link between A21Asn and B29Lys. It was analyzed using static and dynamic light scattering and small-angle X-ray scattering to evaluate its self-association behavior. The tertiary structure was obtained using nuclear magnetic resonance and X-ray crystallography. The biological activity of HMWP was determined using 2 in vitro assays, and its influence on fibrillation was investigated using Thioflavin T assays. The dimer's tertiary structure was nearly identical to that of the noncovalent insulin dimer, and it was able to form hexamers in the presence of zinc. The dimer exhibited reduced propensity for self-association in the absence of zinc but significantly postponed the onset of fibrillation in insulin formulations. Consistent with its dimeric state, the tested species of HMWP showed little to no biological activity in the used assays. This study is the first detailed characterization of a specific type of human insulin HMWP formed during storage of a marketed pharmaceutical formulation. These results indicate that this specific type of HMWP is unlikely to antagonize the physical stability of the formulation, as HMWP retained a tertiary structure similar to the noncovalent dimer and participated in hexamer assembly in the presence of zinc. In addition, increasing amounts of HMWP reduce the rate of insulin fibrillation.
Asunto(s)
Hipoglucemiantes/química , Insulina/química , Cristalografía por Rayos X , Almacenaje de Medicamentos , Humanos , Modelos Moleculares , Agregado de Proteínas , Multimerización de Proteína , Estructura Terciaria de Proteína , Zinc/químicaRESUMEN
The monoclonal antibody GSLA-2 recognizes the sialyl Lewis(a) (sLe(a)) epitope, which has an increased occurrence on mucin type glycoproteins of patients with colorectal carcinoma. GSLA-2 is therefore used in tumor diagnosis. To advance the understanding of this highly specific molecular recognition reaction, we have analyzed the binding epitope of sLe(a) at atomic resolution using saturation transfer difference NMR. To compare, the binding epitopes of sialyl Lewis(x) (sLe(x)) and of four synthetic derivatives of sLe(a) were explored. Surface plasmon resonance experiments furnished thermodynamic and kinetic data. It is observed that all pyranose rings of sLe(a) are in contact with the protein surface, with the neuramic acid residue receiving the largest fraction of saturation transfer. In contrast, sLe(x) binds very weakly, though specifically to GSLA-2, with a different binding epitope. This study provides a comprehensive picture of the recognition sLe(a) and explains the exquisite specificity of the antibody.
Asunto(s)
Anticuerpos Monoclonales/química , Antígenos de Carbohidratos Asociados a Tumores/química , Gangliósidos/química , Sitios de Unión , Antígeno CA-19-9 , Secuencia de Carbohidratos , Mapeo Epitopo , Cinética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Oligosacáridos/química , Antígeno Sialil Lewis X , Resonancia por Plasmón de Superficie , TermodinámicaRESUMEN
The siglecs are a family of I-type lectins binding to sialic acids on the cell surface. Sialoadhesin (siglec-1) is expressed at much higher levels in inflammatory macrophages and specifically binds to alpha-2,3-sialylated N-acetyl lactosamine residues of glycan chains. The terminal disaccharide alpha-D-Neu5Ac-(2-->3)-beta-D-Gal is thought to be the main epitope recognized by sialoadhesin. To understand the basis of this biological recognition reaction we combined NMR experiments with a molecular modeling study. We employed saturation transfer difference (STD) NMR experiments to characterize the binding epitope of alpha-2,3-sialylated lactose, alpha-D-Neu5Ac-(2-->3)-beta-D-Gal-(1-->4)-D-Glc 1 to sialoadhesin at atomic resolution. The experimental results were compared to a computational docking model and to X-ray data of a complex of sialyl lactose and sialoadhesin. The data reveal that sialoadhesin mainly recognizes the N-acetyl neuraminic acid and a small part of the galactose moiety of 1. The crystal structure of a complex of sialoadhesin with sialyl lactose 1 was used as a basis for a modeling study using the FlexiDock algorithm. The model generated was very similar to the original crystal structure. Therefore, the X-ray data were used to predict theoretical STD values utilizing the CORCEMA-STD protocol. The good agreement between experimental and theoretical STD values indicates that a combined modeling/STD NMR approach yields a reliable structural model for the complex of sialoadhesin with alpha-D-Neu5Ac-(2-->3)-beta-D-Gal-(1-->4)-D-Glc 1 in aqueous solution.
Asunto(s)
Simulación por Computador , Lactosa/análogos & derivados , Lactosa/química , Lactosa/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Receptores Inmunológicos/química , Receptores Inmunológicos/metabolismo , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Secuencia de Carbohidratos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Unión Proteica , Lectina 1 Similar a Ig de Unión al Ácido SiálicoAsunto(s)
Epítopos/química , Gangliósidos/química , Lectinas/química , Glicoproteína Asociada a Mielina/antagonistas & inhibidores , Glicoproteína Asociada a Mielina/química , Conformación de Carbohidratos , Simulación por Computador , Selectina E/química , Selectina E/farmacología , Epítopos/metabolismo , Gangliósidos/farmacología , Glicoproteína Asociada a Mielina/metabolismo , Resonancia Magnética Nuclear Biomolecular , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Obesity and type 2 diabetes have reached epidemic proportions; however, scarce information about how these metabolic syndromes influence brain energy and neurotransmitter homeostasis exist. The objective of this study was to elucidate how brain glycogen and neurotransmitter homeostasis are affected by these conditions. [1-(13)C]glucose was administered to Zucker obese (ZO) and Zucker diabetic fatty (ZDF) rats. Sprague-Dawley (SprD), Zucker lean (ZL), and ZDF lean rats were used as controls. Several brain regions were analyzed for glycogen levels along with (13)C-labeling and content of glutamate, glutamine, GABA, aspartate, and alanine. Blood glucose concentrations and (13)C enrichment were determined. (13)C-labeling in glutamate was lower in ZO and ZDF rats in comparison with the controls. The molecular carbon labeling (MCL) ratio between alanine and glutamate was higher in the ZDF rats. The MCL ratios of glutamine and glutamate were decreased in the cerebellum of the ZO and the ZDF rats. Glycogen levels were also lower in this region. These results suggest that the obese and type 2 diabetic models were associated with lower brain glucose metabolism. Glucose metabolism through the TCA cycle was more decreased than glycolytic activity. Furthermore, reduced glutamate-glutamine cycling was also observed in the obese and type 2 diabetic states.
Asunto(s)
Aminoácidos/metabolismo , Encéfalo/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glucógeno/metabolismo , Obesidad/metabolismo , Alanina/metabolismo , Animales , Ácido Aspártico/metabolismo , Glucemia/metabolismo , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Ratas Zucker , Ácido gamma-Aminobutírico/metabolismoRESUMEN
To generate a library covering a relatively wide area of the chemical space, molecular descriptors, and multivariate data analysis were combined to select the building blocks required for generating a diversity optimized library of putative Fc-fragment binding ligands. In such a method of library design, structural information about the target protein is not needed. Synthesis of the resulting 770 member virtual library was carried out using encoded beads, which facilitated rapid identification of the subsequent hits. The library was screened in an on-bead fluorescence assay with immunoglobulin G Fc-fragment of the subtype 4 to identify Fc-fragment binding ligands that would be useful for purifying monoclonal antibodies. An analysis of the positions of the hits in the chemical space revealed that the ones with the highest fluorescence were primarily concentrated in a particular part of the chemical space. One of the identified hits, when immobilized on amino sepharose, was able to purify a monoclonal antibody from crude cell supernatant with purity of 84% and a 70% recovery. The chemometric tools employed for the library design allowed the identification of the fraction of the available chemical space that would be preferred for a second generation library.
Asunto(s)
Fragmentos Fc de Inmunoglobulinas/química , Técnicas Químicas Combinatorias/clasificación , Técnicas Químicas Combinatorias/métodos , Bases de Datos de Proteínas , Cromatografía de Gases y Espectrometría de Masas , Ligandos , Estructura Molecular , Péptidos/análisis , Proteómica/métodosRESUMEN
3D H2BC is introduced for heteronuclear assignment on natural abundance samples even for biomolecules up to at least 10 kDa in low millimolar concentrations as an overnight experiment using the latest generation of cryogenically cooled probes. The short pulse sequence duration of H2BC is maintained in the 3D version due to multiple use of the constant-time delay. Applications ranging from a small lipid to a non-recombinant protein demonstrate the merits of 3D H2BC and the ease of obtaining assignments in chains of protonated carbons.
Asunto(s)
Algoritmos , Biopolímeros/análisis , Biopolímeros/química , Isótopos/análisis , Isótopos/química , Modelos Químicos , Resonancia Magnética Nuclear Biomolecular/métodos , Simulación por Computador , Sensibilidad y EspecificidadRESUMEN
Hexokinase catalyzes the phosphorylation of glucose and is the first enzyme in glycolysis. To investigate enzyme-ligand interactions in yeast hexokinase isoform PII under physiological conditions, we utilized the technique of Saturation Transfer Difference NMR (STD NMR) to monitor binding modes and binding affinities of different ligands at atomic resolution. These experiments clearly show that hexokinase tolerates several changes at C-2 of its main substrate glucose, whereas epimerization of C-4 significantly reduces ligand binding. Although both glucose anomers bind to yeast hexokinase, the alpha-form is the preferred form for the phosphorylation reaction. These findings allow mapping of tolerated and prohibited modification sites on the ligand. Furthermore, competitive titration experiments show that mannose has the highest binding affinity of all examined sugars. As several naturally occurring sugars in cells show binding affinities in a similar range, hexokinase may be considered as an 'emergency enzyme' in yeast cells. Taken together, our results represent a comprehensive analysis of ligand-enzyme interactions in hexokinase PII and provide a valuable basis for inhibitor design and metabolic engineering.
Asunto(s)
Hexoquinasa/química , Hexoquinasa/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Levaduras/enzimología , Estructura Molecular , Especificidad por SustratoRESUMEN
A new improved multiplicity-edited HMBC experiment is introduced that leads to better J cross-talk suppression in the even (i.e. C + CH2 groups) and odd (i.e. CH + CH3 groups) subspectra. By combining data recorded with three different pulse sequences J cross-talk becomes a second-order effect in Delta1J, i.e. the deviation of an actual 1J coupling constant from the value 1J0 used in setting delays tau = (1J0)(-1/2), which is adequate for most applications. As for the original multiplicity-edited HMBC experiment, the improved experiment can be performed with a single excitation delay or implemented in a broadband version similar to broadband HMBC.