RESUMEN
Constitutive heterozygous GATA2 mutation is associated with deafness, lymphedema, mononuclear cytopenias, infection, myelodysplasia (MDS), and acute myeloid leukemia. In this study, we describe a cross-sectional analysis of 24 patients and 6 relatives with 14 different frameshift or substitution mutations of GATA2. A pattern of dendritic cell, monocyte, B, and natural killer (NK) lymphoid deficiency (DCML deficiency) with elevated Fms-like tyrosine kinase 3 ligand (Flt3L) was observed in all 20 patients phenotyped, including patients with Emberger syndrome, monocytopenia with Mycobacterium avium complex (MonoMAC), and MDS. Four unaffected relatives had a normal phenotype indicating that cellular deficiency may evolve over time or is incompletely penetrant, while 2 developed subclinical cytopenias or elevated Flt3L. Patients with GATA2 mutation maintained higher hemoglobin, neutrophils, and platelets and were younger than controls with acquired MDS and wild-type GATA2. Frameshift mutations were associated with earlier age of clinical presentation than substitution mutations. Elevated Flt3L, loss of bone marrow progenitors, and clonal myelopoiesis were early signs of disease evolution. Clinical progression was associated with increasingly elevated Flt3L, depletion of transitional B cells, CD56(bright) NK cells, naïve T cells, and accumulation of terminally differentiated NK and CD8(+) memory T cells. These studies provide a framework for clinical and laboratory monitoring of patients with GATA2 mutation and may inform therapeutic decision-making.
Asunto(s)
Linfocitos B/patología , Células Dendríticas/patología , Factor de Transcripción GATA2/genética , Células Asesinas Naturales/patología , Monocitos/patología , Mutación/genética , Síndromes Mielodisplásicos/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Linfocitos B/inmunología , Linfocitos B/metabolismo , Biomarcadores , Estudios de Casos y Controles , Niño , Preescolar , Evolución Clonal , Estudios Transversales , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Estudios de Seguimiento , Estudios de Asociación Genética , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Masculino , Persona de Mediana Edad , Monocitos/inmunología , Monocitos/metabolismo , Síndromes Mielodisplásicos/sangre , Síndromes Mielodisplásicos/genética , Linaje , Pronóstico , Adulto Joven , Tirosina Quinasa 3 Similar a fms/metabolismoRESUMEN
Clonality can be established by a lack of mosaicism in a female because of random inactivation of either the maternal or paternal X chromosome early in embryogenesis. The methylation status of CpG sites close to the trinucleotide repeats in exon 1 of the human androgen receptor (AR) X chromosome gene assay (HUMARA) has been used to determine clonality. This HUMARA at times indicated clonal hematopoiesis in healthy elderly women, thus precluding its applicability. We used a clonality assay based on quantitative expression of polymorphic X chromosome genes (qTCA) and found no evidence of clonal hematopoiesis in healthy nonanemic elderly persons. We found instances of discordance between HUMARA results and those obtained by pyrosequencing and qTCA methods, as well as by directly quantifying AR gene expression. To determine the basis of this discrepancy we examined the methylation pattern of the AR locus subject to HUMARA. Notably, we found the extent of DNA methylation to be highly variable at the AR gene in granulocytes of persons with discordant results and also in erythroid burst-forming unit colonies but not in those with clonal hematopoiesis. These data provide the molecular basis of incomplete correlation with the pattern of DNA methylation of this X chromosome AR gene locus.
Asunto(s)
Metilación de ADN , Receptores Androgénicos/genética , Inactivación del Cromosoma X/genética , Adulto , Estudios de Casos y Controles , Cromosomas Humanos X/genética , Cromosomas Humanos X/metabolismo , Evolución Clonal/genética , Metilación de ADN/fisiología , Femenino , Frecuencia de los Genes , Sitios Genéticos , Genotipo , Humanos , Janus Quinasa 2/genética , Masculino , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/metabolismo , Polimorfismo Genético , Receptores Androgénicos/metabolismo , Análisis de Secuencia de ADN/métodos , Repeticiones de Trinucleótidos/genética , Inactivación del Cromosoma X/fisiologíaAsunto(s)
Calreticulina/genética , Leucemia Linfocítica Crónica de Células B/complicaciones , Mielofibrosis Primaria/complicaciones , Mielofibrosis Primaria/genética , Anciano , Células Clonales/patología , Humanos , Leucemia Linfocítica Crónica de Células B/diagnóstico , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Mutación , Mielofibrosis Primaria/patología , Trombocitosis/complicaciones , Trombocitosis/genética , Trombocitosis/patologíaRESUMEN
TET2 mutations are found in polycythemia vera and it was initially reported that there is a greater TET2 mutational burden than JAK2(V617F) in polycythemia vera stem cells and that TET2 mutations precede JAK2(V617F). We quantified the proportion of TET2, JAK2(V617F) mutations and X-chromosome allelic usage in polycythemia vera cells, BFU-Es and in vitro expanded erythroid progenitors and found clonal reticulocytes, granulocytes, platelets and CD34(+) cells. We found that TET2 mutations may also follow rather than precede JAK2(V617F) as recently reported by others. Only a fraction of clonal early hematopoietic precursors and largely polyclonal T cells carry the TET2 mutation. We showed that in vitro the concomitant presence of JAK2(V617F) and TET2 mutations favors clonal polycythemia vera erythroid progenitors in contrast with non-TET2 mutated progenitors. We conclude that loss-of-function TET2 mutations are not the polycythemia vera initiating events and that the acquisition of TET2 somatic mutations may increase the aggressivity of the polycythemia vera clone.
Asunto(s)
Proteínas de Unión al ADN/genética , Janus Quinasa 2/genética , Mutación , Policitemia Vera/genética , Proteínas Proto-Oncogénicas/genética , Sustitución de Aminoácidos , Plaquetas/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cromosomas Humanos X/genética , Células Clonales/metabolismo , Dioxigenasas , Células Precursoras Eritroides/metabolismo , Eritropoyetina/farmacología , Femenino , Citometría de Flujo , Granulocitos/metabolismo , Sistema Hematopoyético/metabolismo , Humanos , Reticulocitos/metabolismo , Linfocitos T/metabolismo , Factores de TiempoRESUMEN
Clonality assays, based on X-chromosome inactivation, discriminate active from inactive alleles. Skewing of X-chromosome allelic usage, based on preferential methylation of one of the HUMARA alleles, was reported as evidence of clonal hematopoiesis in approximately 30% of elderly women. Using a quantitative, transcriptionally based clonality assay, we reported X-chromosome-transcribed allelic ratio in blood cells of healthy women consistent with random X-inactivation of 8 embryonic hematopoietic stem cells. Furthermore, we did not detect clonal hematopoiesis in more than 200 healthy nonelderly women. In view of the susceptibility of aging hematopoietic stem cells to epigenetic dysregulation, we reinvestigated the issue of clonality in elderly women. Forty healthy women (ages 65-92 years; mean, 81.3 years) were tested by a novel, quantitative polymerase chain reaction (qPCR) transcriptional clonality assay. We did not detect clonal hematopoiesis in any of the tested subjects. We also tested DNA from the same granulocyte samples using the methylation-based HUMARA assay, and confirmed previous reports of approximately 30% extensively skewed or monoallelic methylation, in agreement with likely age-related deregulated methylation of the HUMARA gene locus. We conclude that the transcriptionally based X-chromosome clonality assays are suitable for evaluation of clonal hematopoiesis in elderly women.
Asunto(s)
Cromosomas Humanos X , Hematopoyesis , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Estudios de Casos y Controles , Metilación de ADN , Femenino , Humanos , Fenotipo , Polimorfismo de Nucleótido Simple , Receptores Androgénicos/genética , Transcripción Genética , Inactivación del Cromosoma XRESUMEN
PURPOSE: Myelofibrosis is a hematopoietic stem cell neoplasm characterized by bone marrow reticulin fibrosis, extramedullary hematopoiesis, and frequent transformation to acute myeloid leukemia. Constitutive activation of JAK/STAT signaling through mutations in JAK2, CALR, or MPL is central to myelofibrosis pathogenesis. JAK inhibitors such as ruxolitinib reduce symptoms and improve quality of life, but are not curative and do not prevent leukemic transformation, defining a need to identify better therapeutic targets in myelofibrosis. EXPERIMENTAL DESIGN: A short hairpin RNA library screening was performed on JAK2V617F-mutant HEL cells. Nuclear-cytoplasmic transport (NCT) genes including RAN and RANBP2 were among top candidates. JAK2V617F-mutant cell lines, human primary myelofibrosis CD34+ cells, and a retroviral JAK2V617F-driven myeloproliferative neoplasms mouse model were used to determine the effects of inhibiting NCT with selective inhibitors of nuclear export compounds KPT-330 (selinexor) or KPT-8602 (eltanexor). RESULTS: JAK2V617F-mutant HEL, SET-2, and HEL cells resistant to JAK inhibition are exquisitely sensitive to RAN knockdown or pharmacologic inhibition by KPT-330 or KPT-8602. Inhibition of NCT selectively decreased viable cells and colony formation by myelofibrosis compared with cord blood CD34+ cells and enhanced ruxolitinib-mediated growth inhibition and apoptosis, both in newly diagnosed and ruxolitinib-exposed myelofibrosis cells. Inhibition of NCT in myelofibrosis CD34+ cells led to nuclear accumulation of p53. KPT-330 in combination with ruxolitinib-normalized white blood cells, hematocrit, spleen size, and architecture, and selectively reduced JAK2V617F-mutant cells in vivo. CONCLUSIONS: Our data implicate NCT as a potential therapeutic target in myelofibrosis and provide a rationale for clinical evaluation in ruxolitinib-exposed patients with myelofibrosis.
Asunto(s)
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Mielofibrosis Primaria/metabolismo , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Transporte Biológico/efectos de los fármacos , Biomarcadores , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Biología Computacional/métodos , Citoplasma/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Quinasas Janus/genética , Quinasas Janus/metabolismo , Ratones , Terapia Molecular Dirigida , Mutación , Trastornos Mieloproliferativos/etiología , Trastornos Mieloproliferativos/metabolismo , Trastornos Mieloproliferativos/patología , Mielofibrosis Primaria/tratamiento farmacológico , Mielofibrosis Primaria/etiología , Factores de Transcripción STAT/metabolismo , TranscriptomaRESUMEN
To gain insight into the role of the strictly conserved histidine residue, H79, in the reaction mechanism of the methionyl aminopeptidase from Escherichia coli ( EcMetAP-I), the H79A mutated enzyme was prepared. Co(II)-loaded H79A exhibits an overall >7000-fold decrease in specific activity. The almost complete loss of activity is primarily due to a >6000-fold decrease in k cat. Interestingly, the K m value obtained for Co(II)-loaded H79A was approximately half the value observed for wild-type (WT) EcMetAP-I. Consequently, k cat/ K m values decreased only 3000-fold. On the other hand, the observed specific activity of Mn(II)-loaded H79A EcMetAP-I decreased by approximately 2.6-fold while k cat decreased by approximately 3.5-fold. The observed K m value for Mn(II)-loaded H79A EcMetAP-I was approximately 1.4-fold larger than that observed for WT EcMetAP-I, resulting in a k cat/ K m value that is lower by approximately 3.4-fold. Metal binding, UV-vis, and EPR data indicate that the active site is unperturbed by mutation of H79, as suggested by X-ray crystallographic data. Kinetic isotope data indicate that H79 does not transfer a proton to the newly forming amine since a single proton is transferred in the transition state for both the WT and H79A EcMetAP-I enzymes. Therefore, H79 functions to position the substrate by hydrogen bonding to either the amine group of the peptide linkage or a backbone carbonyl group. Together, these data provide new insight into the catalytic mechanism of EcMetAP-I.
Asunto(s)
Aminopeptidasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Histidina/metabolismo , Aminopeptidasas/química , Aminopeptidasas/genética , Sitios de Unión/genética , Catálisis , Cobalto/química , Cobalto/metabolismo , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Histidina/química , Histidina/genética , Hierro/química , Hierro/metabolismo , Cinética , Metionil Aminopeptidasas , Modelos Moleculares , Mutagénesis Sitio-DirigidaRESUMEN
OBJECTIVE: The somatic JAK2(V617F) mutation is seen in most polycythemia vera (PV) patients; however, it is not clear if JAK2(V617F) is the PV-initiating mutation. METHODS: In order to examine this issue, we developed a novel real-time quantitative allele-specific PCR, in which allelic discrimination is enhanced by the synergistic effect of a mismatch in the -1 position, and a locked nucleic acid (LNA) nucleoside at the -2 position. RESULTS: Determination of allelic frequencies was reproducible (SD = 1.5%) and sensitive--0.1% mutant allele detected in 40 ng of DNA. The JAK2(V617F) frequency in clonal granulocytes from 3 PV females was less than 50% (27.5 +/- 11) and in 7 females greater than 50% (75 +/- 10.5). We also found that wild-type JAK2 BFU-E colonies from PV patients can grow without erythropoietin. The identification of the primary genetic lesion resulting in PV is essential for the development of novel therapeutic strategies. CONCLUSION: Our studies correlating the frequency of JAK2(V617F) mutant allele and clonality, as well as the presence of homozygous wild-type JAK2 erythropoietin-independent erythroid colonies, provide compelling evidence that the JAK2(V617F) is not the PV-initiating mutation. This supports a model wherein the JAK2(V617F) mutation arises as a secondary genetic event. Furthermore, our results indicate that an undefined molecular lesion, preceding JAK2(V617F), is responsible for clonal hematopoiesis in PV. We conclude that development of therapeutic strategies that target the JAK2(V617F) clonal cells may not be sufficient for eradication of PV.
Asunto(s)
Janus Quinasa 2/genética , Mutación Missense/fisiología , Policitemia Vera/etiología , Policitemia Vera/genética , Células Clonales/patología , Sondas de ADN/normas , Eritropoyetina/farmacología , Femenino , Frecuencia de los Genes , Humanos , Policitemia Vera/patología , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa/normasAsunto(s)
Janus Quinasa 2/sangre , Janus Quinasa 2/genética , Mutación , Trastornos Mieloproliferativos/sangre , Trastornos Mieloproliferativos/genética , Conservación de la Sangre , Separación Celular , ADN/sangre , ADN/genética , Análisis Mutacional de ADN , Granulocitos/enzimología , Humanos , Trastornos Mieloproliferativos/enzimología , ARN Mensajero/sangre , ARN Mensajero/genética , Factores de TiempoAsunto(s)
Envejecimiento/genética , Cromosomas Humanos X/genética , Hematopoyesis/genética , Neutrófilos/fisiología , Reacción en Cadena de la Polimerasa/métodos , Inactivación del Cromosoma X/genética , Adulto , Anciano , Anciano de 80 o más Años , Metilación de ADN , Compensación de Dosificación (Genética) , Femenino , Humanos , Persona de Mediana Edad , Modelos Genéticos , Reacción en Cadena de la Polimerasa/normas , Polimorfismo de Nucleótido Simple , Reproducibilidad de los ResultadosRESUMEN
Understanding the reaction mechanism of co-catalytic metallopeptidases provides a starting point for the design and synthesis of new molecules that can be screened as potential pharmaceuticals. Many of the enzymes that contain co-catalytic metallo-active sites play important roles in cellular processes such as tissue repair, protein maturation, hormone level regulation, cell-cycle control and protein degradation. Therefore, these enzymes play central roles in several disease states including cancer, HIV, stroke, diabetes, bacterial infections, neurological processes, schizophrenia, seizure disorders, and amyotrophic lateral sclerosis. The mechanism of AAP, an aminopeptidase from Aeromonas proteolytica, is one of the best-characterized examples of a metallopeptidase containing a co-catalytic metallo-active site, although this enzyme is not a specific pharmaceutical target at this time. As a large majority of co-catalytic metallopeptidases contain active sites that are nearly identical to the one observed in AAP, the major steps of their catalytic mechanisms are likely to be very similar. With this in mind, it is possible to propose a general catalytic mechanism for the hydrolysis of amino acid substrates.
Asunto(s)
Metaloendopeptidasas/metabolismo , Amidohidrolasas/antagonistas & inhibidores , Amidohidrolasas/metabolismo , Aminopeptidasas/antagonistas & inhibidores , Aminopeptidasas/metabolismo , Animales , Carboxipeptidasas/antagonistas & inhibidores , Carboxipeptidasas/metabolismo , Catálisis , Humanos , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/efectos de los fármacos , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacologíaRESUMEN
Interferon α (IFNα) is used clinically to restore polyclonal hematopoiesis in patients with the myeloproliferative neoplasms polycythemia vera and essential thrombocythemia and to improve chemosensitivity in chronic myeloid leukemia patients. However, the mechanisms by which IFNα affects disease-initiating hematopoietic stem and progenitor cells (HSPCs) remain poorly understood. Although IFNα has been found to transiently impair quiescence of murine hematopoietic stem cells, its effects on human HSPCs have not been studied in vivo. Here, we compared bone marrow serially obtained from patients with myeloproliferative neoplasms before and during pegylated IFNα treatment against marrow serially obtained from patients on hydroxyurea. The percentage of HSPCs actively undergoing cell cycle was increased after pegylated IFNα treatment in a majority of patients compared with hydroxyurea-treated controls, suggesting that IFNα promotes cell division. Furthermore, transcriptional profiling revealed that cell cycle-associated genes were induced, whereas genes involved in HSPC quiescence were repressed during IFNα treatment. Compared with hydroxyurea-treated controls, pegylated IFNα-treated patients had similar numbers of HSPCs, but increased numbers of hematopoietic progenitors as determined by colony formation assay, indicating an increase in myeloid proliferation/differentiation. These effects occurred regardless of JAK2 mutational status. Together, these data provide the first in vivo evidence that pegylated IFNα promotes cell division and differentiation of human HSPCs.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Hidroxiurea/administración & dosificación , Interferón-alfa/administración & dosificación , Leucemia Mielógena Crónica BCR-ABL Positiva , Policitemia Vera , Trombocitemia Esencial , Anciano , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Femenino , Células Madre Hematopoyéticas/patología , Humanos , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Masculino , Ratones , Persona de Mediana Edad , Policitemia Vera/tratamiento farmacológico , Policitemia Vera/genética , Policitemia Vera/metabolismo , Policitemia Vera/patología , Trombocitemia Esencial/tratamiento farmacológico , Trombocitemia Esencial/genética , Trombocitemia Esencial/metabolismo , Trombocitemia Esencial/patología , Factores de TiempoRESUMEN
The H355A, H355K, H80A, and H80K mutant enzymes of the argE-encoded N-acetyl-L-ornithine deacetylase (ArgE) from Escherichia coli were prepared, however, only the H355A enzyme was found to be soluble. Kinetic analysis of the Co(II)-loaded H355A exhibited activity levels that were 380-fold less than Co(II)-loaded WT ArgE. Electronic absorption spectra of Co(II)-loaded H355A-ArgE indicate that the bound Co(II) ion resides in a distorted, five-coordinate environment and Isothermal Titration Calorimetry (ITC) data for Zn(II) binding to the H355A enzyme provided a dissociation constant (K d) of 39 µM. A three-dimensional homology model of ArgE was generated using the X-ray crystal structure of the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae confirming the assignment of H355 as well as H80 as active site ligands.
RESUMEN
The catalytically competent Mn(II)-loaded form of the argE-encoded N-acetyl-L-ornithine deacetylase from Escherichia coli (ArgE) was characterized by kinetic, thermodynamic, and spectroscopic methods. Maximum N-acetyl-L-ornithine (NAO) hydrolytic activity was observed in the presence of one Mn(II) ion with k(cat) and K(m) values of 550 s(-1) and 0.8 mM, respectively, providing a catalytic efficiency (k(cat)/K(m)) of 6.9 x 10(5) M(-1) s(-1). The ArgE dissociation constant (K(d)) for Mn(II) was determined to be 0.18 microM, correlating well with a value obtained by isothermal titration calorimetry of 0.30 microM for the first metal binding event and 5.3 microM for the second. An Arrhenius plot of the NAO hydrolysis for Mn(II)-loaded ArgE was linear from 15 to 55 degrees C, suggesting the rate-limiting step does not change as a function of temperature over this range. The activation energy, determined from the slope of this plot, was 50.3 kJ mol(-1). Other thermodynamic parameters were DeltaG(double dagger) = 58.1 kJ mol(-1), DeltaH(double dagger) = 47.7 kJ mol(-1), and DeltaS(double dagger) = -34.5 J mol(-1) K(-1). Similarly, plots of lnK(m) versus 1/T were linear, suggesting substrate binding is controlled by a single step. The natural product, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]leucine (bestatin), was found to be a competitive inhibitor of ArgE with a K (i) value of 67 muM. Electron paramagnetic resonance (EPR) data recorded for both [Mn(II)_(ArgE)] and [Mn(II)Mn(II)(ArgE)] indicate that the two Mn(II) ions form a dinuclear site. Moreover, the EPR spectrum of [Mn(II)Mn(II)(ArgE)] in the presence of bestatin indicates that bestatin binds to ArgE but does not form a micro-alkoxide bridge between the two metal ions.
Asunto(s)
Amidohidrolasas/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Genes Bacterianos/genética , Manganeso/metabolismo , Amidohidrolasas/antagonistas & inhibidores , Amidohidrolasas/biosíntesis , Apoenzimas/metabolismo , Calorimetría , Catálisis , Espectroscopía de Resonancia por Spin del Electrón , Inhibidores Enzimáticos/farmacología , Hidrólisis , Cinética , Leucina/análogos & derivados , Leucina/farmacología , Inhibidores de Proteasas/farmacología , Espectrofotometría Ultravioleta , TermodinámicaRESUMEN
Glutamate-134 (E134) is proposed to act as the general acid/base during the hydrolysis reaction catalyzed by the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae. To date, no direct evidence has been reported for the role of E134 during catalytic turnover by DapE. In order to elucidate the catalytic role of E134, altered DapE enzymes were prepared in which E134 was substituted with an alanine and an aspartate residue. The Michaelis constant (K (m)) does not change upon substitution with aspartate but the rate of the reaction changes drastically in the following order: glutamate (100% activity), aspartate (0.09%), and alanine (0%). Examination of the pH dependence of the kinetic constants k (cat) and K (m) for E134D-DapE revealed ionizations at pH 6.4, 7.4, and approximately 9.7. Isothermal titration calorimetry experiments revealed a significant weakening in metal K (d) values of E134D-DapE. D134 and A134 perturb the second divalent metal binding site significantly more than the first, but both altered enzymes can still bind two divalent metal ions. Structural perturbations of the dinuclear active site of DapE were also examined for two E134-substituted forms, namely E134D-DapE and E134A-DapE, by UV-vis and electron paramagnetic resonance (EPR) spectroscopy. UV-vis spectroscopy of Co(II)-substituted E134D-DapE and E134A-DapE did not reveal any significant changes in the electronic absorption spectra, suggesting that both Co(II) ions in E134D-DapE and E134A-DapE reside in distorted trigonal bipyramidal coordination geometries. EPR spectra of [Co_(E134D-DapE)] and [Co_(E1341A-DapE] are similar to those observed for [CoCo(DapE)] and somewhat similar to the spectrum of [Co(H(2)O)(6)](2+) which typically exhibit E/D values of approximately 0.1. Computer simulation returned an axial g-tensor with g ((x,y))=2.24 and E/D=0.07; g ( z ) was only poorly determined, but was estimated as 2.5-2.6. Upon the addition of a second Co(II) ion to [Co_(E134D-DapE)] and [Co_(E134A-DapE)], a broad axial signal was observed; however, no signals were observed with B (0)||B (1) ("parallel mode"). On the basis of these data, E134 is intrinsically involved in the hydrolysis reaction catalyzed by DapE and likely plays the role of a general acid and base.
Asunto(s)
Amidohidrolasas/química , Amidohidrolasas/metabolismo , Ácido Diaminopimélico/metabolismo , Haemophilus influenzae/enzimología , Amidohidrolasas/genética , Sitios de Unión , Calorimetría , Espectroscopía de Resonancia por Spin del Electrón , Activación Enzimática , Concentración de Iones de Hidrógeno , Cinética , Metales/farmacología , Estructura Molecular , Mutagénesis Sitio-Dirigida , Análisis EspectralRESUMEN
Previous kinetic characterization of the glutamate 151 (E151)-substituted forms of the leucine aminopeptidase from Aeromonas proteolytica (Vibrio proteolyticus; AAP) has provided critical evidence that this residue functions as the general acid/base. The close proximity of similar glutamate residues to the bridging water/hydroxide of the dinuclear active sites of metalloenzymes (2.80 and 3.94 angstroms in carboxypeptidase G2 and 3.30 and 3.63 angstroms in AAP), suggests it may also be involved in stabilizing the active-site metal ions. Therefore, the structural perturbations of the dinuclear active site of AAP were examined for two E151-substituted forms, namely E151D-AAP and E151A-AAP, by UV-vis and electron paramagnetic resonance (EPR) spectroscopy. UV-vis spectroscopy of Co(II)-substituted E151A-AAP did not reveal any significant changes in the electronic absorption spectra. However UV-vis spectra of mono- and dicobalt(II) E151D-AAP exhibited a lower molecular absorptivity compared to AAP (23 and 43 M(-1) cm(-1) vs. 56 and 109 M(-1) cm(-1) for E151D-AAP and AAP, respectively) suggesting both Co(II) ions reside in distorted octahedral coordination geometry in E151D-AAP. EPR spectra of [Co_(E151D-AAP)], [ZnCo(E151D-AAP)], and [(CoCo(E151D-AAP)] were identical, with g(perpendicular) = 2.35, g(parallel) = 2.19, and E/D = 0.19, similar to [CoCo(AAP)]. On the other hand, the EPR spectrum of [Co_(E151A-AAP)] was best simulated assuming the presence of two species with (i) g(x,y) = 2.509, g(z) = 2.19, E/D = 0.19, A = 0.0069 cm(-1) and (ii) g(x,y) = 2.565, g(z) = 2.19, E/D = 0.20, A = 0.0082 cm(-1) indicative of a five- or six-coordinate species. Isothermal titration calorimetry experiments revealed a large decrease in Zn(II) affinities, with K(d) values elevated by factors of approximately 850 and approximately 24,000 for the first metal binding events of E151D- and E151A-AAP, respectively. The combination of these data indicates that E151 serves to stabilize the dinuclear active site of AAP.
Asunto(s)
Aminopeptidasas/química , Aminopeptidasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Aeromonas/enzimología , Sitios de Unión , Calorimetría , Cobalto/química , Cobalto/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Ácido Glutámico/química , Ácido Glutámico/metabolismo , Enlace de Hidrógeno , Cinética , Mutación Puntual , Espectrofotometría Ultravioleta , Zinc/química , Zinc/metabolismoRESUMEN
Metalloproteases utilize their active site divalent metal ions to generate a nucleophilic water/hydroxide. For methionine aminopeptidases (MetAPs), the exact location of this nucleophile, as well as of the substrate, with respect to the active site metal ion is unknown. In order to address this issue, we have examined the catalytically competent Fe(II)-loaded form of PfMetAP-II ([Fe(PfMetAP-II)]) in the absence and presence of both nitric oxide (NO) and the substrate-analogue inhibitor butaneboronic acid (BuBA) by kinetic and spectroscopic (EPR, UV-vis) methods. NO binds to [Fe(PfMetAP-II)] with a Kd of 200 microM forming an {FeNO}7 complex. UV-vis spectra of the resulting [Fe(PfMetAP-II)]-NO complex indicate that the Fe(II) ion is six coordinate. These data suggest that NO binding occurs without displacing the bound aquo/hydroxo moiety in [Fe(PfMetAP-II)]. On the basis of EPR spectra, the resulting Fe-NO complex is best described as NO- (S = 1) antiferromagnetically coupled to a high-spin Fe(III) ion (S = 5/2). The addition of BuBA to [Fe(PfMetAP-II)]-NO displaces the coordinated water molecule forming a six-coordinate adduct. EPR data also indicate that an interaction between the bound NO- and BuBA occurs forming a complex that mimics an intermediate step between the Michaelis complex and the tetrahedral transition-state.
Asunto(s)
Aminopeptidasas/química , Hierro/química , Metaloendopeptidasas/química , Pyrococcus furiosus/enzimología , Aminopeptidasas/metabolismo , Sitios de Unión , Compuestos de Boro , Dominio Catalítico , Espectroscopía de Resonancia por Spin del Electrón , Cinética , Metaloendopeptidasas/metabolismo , Óxido Nítrico , Análisis Espectral/métodosRESUMEN
Two residues that are conserved in type-I methionyl aminopeptidases (MetAPs) but are absent in all type-II MetAPs are the cysteine residues (Escherichia coli MetAP-I: C59 and C70) that reside at the back of the substrate recognition pocket. These Cys residues are 4.4 A apart and do not form a disulfide bond. Since bacteria and fungi contain only type-I MetAPs while all human cells contain both type-I and type-II MetAPs, type-I MetAPs represent a novel antibiotic/antifungal target if type-I MetAPs can be specifically targeted over type-II. Based on reaction of the thiol-specific binding reagent 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) with the type-I MetAP from E. coli and the type-II MetAP from Pyrococcus furiosus, the type-I MetAP can be selectively inhibited. Verification that DTNB covalently binds to C59 in EcMetAP-I was obtained by mass spectrometry (MS) from reaction of DTNB with the C59A and C70A mutant EcMetAP-I enzymes. In addition, two inhibitors of EcMetAP-I, 5-iodopentaphosphonic acid (1) and 6-phosphonohexanoic acid (2), were designed and synthesized. The first was designed as a selective-C59 binding reagent while the second was designed as a simple competitive inhibitor of EcMetAP. Indeed, inhibitor 1 forms a covalent interaction with C59 based on activity assays and MS measurements, while 2 does not. These data indicate that type-I MetAPs can be selectively targeted over type-II MetAPs, suggesting that type-I MetAPs represent a new enzymatic target for antibacterial or antifungal agents.
Asunto(s)
Aminopeptidasas/clasificación , Aminopeptidasas/metabolismo , Secuencia de Aminoácidos , Aminopeptidasas/química , Aminopeptidasas/genética , Antiinfecciosos/farmacología , Cisteína/genética , Cisteína/metabolismo , Escherichia coli/enzimología , Humanos , Cinética , Metionil Aminopeptidasas , Datos de Secuencia Molecular , Mutación/genética , Pyrococcus furiosus/enzimología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The catalytic and structural properties of the argE-encoded N-acetyl-L-ornithine deacetylase (ArgE) from Escherichia coli were investigated. On the basis of kinetic and ITC (isothermal titration calorimetry) data, Zn(II) binds to ArgE with Kd values that differ by approximately 20 times. Moreover, ArgE exhibits approximately 90% of its full catalytic activity upon addition of one metal ion. Therefore, ArgE behaves similarly to the aminopeptidase from Aeromonas proteolytica (AAP) in that one metal ion is the catalytic metal ion while the second likely plays a structural role. The N-acetyl-L-ornithine (NAO) deacetylase activity of ArgE showed a linear temperature dependence from 20 to 45 degrees C, indicating that the rate-limiting step does not change over this temperature range. The activation energy for NAO hydrolysis by ArgE was 25.6 kJ/mol when loaded with Zn(II) and 34.3 kJ/mol when loaded with Co(II). Electronic absorption and EPR (electron paramagnetic resonance) spectra of [Co x (ArgE)] and [CoCo(ArgE)] indicate that both divalent metal binding sites are five coordinate. In addition, EPR data show clear evidence of spin-spin coupling between the Co(II) ions in the active site but only after addition of a second equivalent of Co(II). Combination of these data provides the first physical evidence that the ArgE from E. coli contains a dinuclear Zn(II) active site, similar to AAP and the carboxypeptidase G2 from Pseudomonas sp. strain RS-16 (CPG2).
Asunto(s)
Amidohidrolasas/química , Cobalto/química , Escherichia coli/enzimología , Compuestos Organometálicos/química , Zinc/química , Amidohidrolasas/genética , Amidohidrolasas/aislamiento & purificación , Sitios de Unión , Catálisis , Conductometría/métodos , Espectroscopía de Resonancia por Spin del Electrón/métodos , Cinética , Temperatura , Factores de TiempoRESUMEN
Glutamate151 (E151) has been shown to be catalytically essential for the aminopeptidase from Vibrio proteolyticus (AAP). E151 acts as the general acid/base during the catalytic mechanism of peptide hydrolysis. However, a glutamate residue is not the only residue capable of functioning as a general acid/base during catalysis for dinuclear metallohydrolases. Recent crystallographic characterization of the D-aminopeptidase from Bacillus subtilis (DppA) revealed a histidine residue that resides in an identical position to E151 in AAP. Because the active-site ligands for DppA and AAP are identical, AAP has been used as a model enzyme to understand the mechanistic role of H115 in DppA. Substitution of E151 with histidine resulted in an active AAP enzyme exhibiting a kcat value of 2.0 min(-1), which is over 2000 times slower than r AAP (4380 min(-1)). ITC experiments revealed that ZnII binds 330 and 3 times more weakly to E151H-AAP compared to r-AAP. UV-vis and EPR spectra of CoII-loaded E151H-AAP indicated that the first metal ion resides in a hexacoordinate/pentacoordinate equilibrium environment, whereas the second metal ion is six-coordinate. pH dependence of the kinetic parameters kcat and K(m) for the hydrolysis of L-leucine p-nitroanilide (L-pNA) revealed a change in an ionization constant in the enzyme-substrate complex from 5.3 in r-AAP to 6.4 in E151H-AAP, consistent with E151 in AAP being the active-site general acid/base. Proton inventory studies at pH 8.50 indicate the transfer of one proton in the rate-limiting step of the reaction. Moreover, the X-ray crystal structure of [ZnZn(E151H-AAP)] has been solved to 1.9 A resolution, and alteration of E151 to histidine does not introduce any major conformational changes to the overall protein structure or the dinuclear ZnII active site. Therefore, a histidine residue can function as the general acid/base in hydrolysis reactions of peptides and, through analogy of the role of E151 in AAP, H115 in DppA likely shuttles a proton to the leaving group of the substrate.