RESUMO
BackgroundUntreated phenylketonuria (PKU), one of the most common human genetic disorders, usually results in mental retardation. Although a protein-restricted artificial diet can prevent retardation, dietary compliance in adults is often poor. In pregnant PKU women, noncompliance can result in maternal PKU syndrome, where high phenylalanine (Phe) levels cause severe fetal complications. Enzyme substitution therapy using Phe ammonia lyase (PAL) corrects PKU in BTBR Phe hydroxylase (Pahenu2) mutant mice, suggesting a potential for maternal PKU syndrome treatment in humans.MethodsWe reviewed clinical data to assess maternal PKU syndrome incidence in pregnant PKU women. We treated female PKU mice (on normal diet) with PAL, stabilizing Phe at physiological levels, and mated them to assess pregnancy outcomes.ResultsPatient records show that, unfortunately, the efficacy of diet to prevent maternal PKU syndrome has not significantly improved since the problem was first noted 40 years ago. PAL treatment of pregnant PKU mice shows that offspring of PAL-treated dams survive to adulthood, in contrast to the complete lethality seen in untreated mice, or limited survival seen in mice on a PKU diet.ConclusionPAL treatment reduced maternal PKU syndrome severity in mice and may have potential for human PKU therapy.
Assuntos
Modelos Animais de Doenças , Fenilalanina Hidroxilase/genética , Fenilcetonúria Materna/genética , Fenilcetonúria Materna/fisiopatologia , Adulto , Amônia-Liases/genética , Animais , Dieta com Restrição de Proteínas , Feminino , Heterozigoto , Homozigoto , Humanos , Deficiência Intelectual/genética , Deficiência Intelectual/fisiopatologia , Camundongos , Camundongos Mutantes , Fenilcetonúria Materna/dietoterapia , Polietilenoglicóis/metabolismo , Gravidez , Resultado da Gravidez , Prenhez , Estudos RetrospectivosRESUMO
Pegylated recombinant phenylalanine ammonia lyase (pegvaliase) is an enzyme substitution therapy being evaluated for the treatment of phenylketonuria (PKU). PKU is characterized by elevated plasma phenylalanine, which is thought to lead to a deficiency in monoamine neurotransmitters and ultimately, neurocognitive dysfunction. A natural history evaluation in a mouse model of PKU demonstrated a profound decrease in tyrosine hydroxylase (TH) immunoreactivity in several brain regions, beginning at 4weeks of age. Following treatment with pegvaliase, the number of TH positive neurons was increased in several brain regions compared to placebo treated ENU2 mice.
Assuntos
Fenilalanina Amônia-Liase/uso terapêutico , Fenilcetonúrias/complicações , Fenilcetonúrias/tratamento farmacológico , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/patologia , Modelos Animais de Doenças , Humanos , Camundongos , Neurotransmissores/administração & dosagem , Neurotransmissores/genética , Neurotransmissores/uso terapêutico , Fenilalanina/sangue , Fenilalanina Amônia-Liase/administração & dosagem , Fenilalanina Amônia-Liase/genética , Fenilcetonúrias/patologia , Fenilcetonúrias/fisiopatologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/uso terapêutico , Tirosina 3-Mono-Oxigenase/imunologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
PURPOSE: Phenylketonuria (PKU) is a disorder of phenylalanine (Phe) metabolism that frequently results in epilepsy if a low Phe diet was not implemented at birth. The mechanisms by which Phe affects the brain are poorly understood. METHODS: Audiogenic seizures (AGS) were studied in female homozygous Pah(enu2) BTBR (PKU) mice. RESULTS: Adult PKU mice, 18-20 weeks of age, in contrast to wild-type and heterozygous counterparts, exhibited a full range of AGS. Younger PKU mice, 5-7 weeks of age, had higher serum Phe levels (2.22 +/- 0.20 mM) in comparison with the adult animals (1.72 +/- 0.05 mM) and were not susceptible to AGS. Among adult mice, animals susceptible to AGS had significantly lower serum Phe levels (1.62 +/- 0.06 mM) in comparison with those resistant to AGS (1.86 +/- 0.07 mM). Susceptibility to AGS tended to increase in the afternoon when serum Phe concentration decreased in comparison to evening and morning. Normalization of serum Phe level by instituting a low Phe diet generally prevented susceptibility to AGS within 12 h. Although return to a standard diet raised Phe levels to hyperphenylalaninemic within 12 h in animals treated with a low Phe diet for 2 weeks, more than 7 weeks were needed for a complete resumption of AGS. CONCLUSIONS: Transient decrease in Phe levels within hyperphenylalaninemic range may be a necessary condition for PKU-related seizures to occur. A low Phe diet prevents susceptibility to seizures, which can resume with the significant delay after termination of dietary treatment.
Assuntos
Epilepsia Reflexa/sangue , Epilepsia/sangue , Fenilalanina/sangue , Fenilcetonúrias/sangue , Estimulação Acústica , Fatores Etários , Animais , Ritmo Circadiano , Modelos Animais de Doenças , Epilepsia/diagnóstico , Epilepsia/genética , Epilepsia Reflexa/diagnóstico , Epilepsia Reflexa/genética , Feminino , Alimentos Formulados , Predisposição Genética para Doença , Camundongos , Camundongos Mutantes , Fenilalanina/administração & dosagem , Fenilalanina/metabolismo , Fenilcetonúrias/dietoterapia , Fenilcetonúrias/genéticaRESUMO
We report the X-ray crystal structures and rate constants for proton transfer in site-specific mutants of human carbonic anhydrase III (HCA III) that place a histidine residue in the active-site cavity: K64H, R67H, and K64H-R67N HCA III. Prior evidence from the exchange of 18O between CO2 and water measured by mass spectrometry shows each mutant to have enhanced proton transfer in catalysis compared with wild-type HCA III. However, His64 in K64H and K64H-R67N HCA III have at most a capacity for proton transfer that is only 13% that of His64 in HCA II. This reduced rate in mutants of HCA III is associated with a constrained side-chain conformation of His64, which is oriented outward, away from the active-site zinc in the crystal structures. This conformation appears stabilized by a prominent pi stacking interaction of the imidazole ring of His64 with the indole ring of Trp5 in mutants of HCA III. This single orientation of His64 in K64H HCA III predominates also in a double mutant K64H-R67N HCA III, indicating that the positive charge of Arg67 does not influence the observed conformation of His64 in the crystal structure. Hence, the structures and catalytic activity of these mutants of HCA III containing His64 account only in small part for the lower activity of this isozyme compared with HCA II. His67 in R67H HCA III was also shown to be a proton shuttle residue, having a capacity for proton transfer that was approximately four times that of His64 in K64H HCA III. This is most likely due to its proximity and orientation inward towards the zinc-bound solvent. These results emphasize the significance of side chain orientation and range of available conformational states as characteristics of an efficient proton shuttle in carbonic anhydrase.
Assuntos
Anidrase Carbônica III/química , Modelos Moleculares , Prótons , Sítios de Ligação/genética , Anidrase Carbônica III/genética , Catálise , Cristalografia por Raios X , Humanos , Cinética , Mutação/genéticaRESUMO
Phenylketonuria (PKU) is a common genetic disorder in humans that arises from deficient activity of phenylalanine hydroxylase (PAH), which catalyzes the conversion of phenylalanine to tyrosine. There is a resultant hyperphenylalanemia with subsequent impairment in cognitive abilities, executive functions and motor coordination. The neuropathogenesis of the disease has not been completely elucidated, however, oxidative stress is considered to be a key feature of the disease process. Hyperphenylalanemia also adversely affects monoaminergic metabolism in the brain. For this reason we chose to evaluate the nigrostriatum of Pah(enu2) mice, to determine if alterations of monoamine metabolism resulted in morphologic nigrostriatal pathology. Furthermore, we believe that recent developments in adeno-associated virus (AAV)-based vectors have greatly increased the potential for long-term gene therapy and may be a viable alternative to dietary treatment for this metabolic disorder. In this study we identified neurodegenerative changes with regenerative responses in the nigrostriatum of Pah(enu2) mice that are consistent with oxidative injury and occurred as early as 4 weeks of age. These neuropathologic changes were reversed following portal vein delivery of a recombinant adeno-associated virus-mouse phenylalanine hydroxylase-woodchuck hepatitis virus post-transcriptional response element (rAAV-mPAH-WPRE) vector to Pah(enu2) mice and corresponded to rapid reduction of serum Phe levels.
Assuntos
Corpo Estriado/patologia , Terapia Genética/métodos , Doenças Neurodegenerativas/patologia , Fenilcetonúrias/patologia , Substância Negra/patologia , Aminoácido Oxirredutases/deficiência , Aminoácido Oxirredutases/genética , Animais , Monoaminas Biogênicas/biossíntese , Corpo Estriado/metabolismo , Corpo Estriado/fisiopatologia , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Terapia Genética/tendências , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Masculino , Camundongos , Camundongos Mutantes Neurológicos , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Degeneração Neural/fisiopatologia , Vias Neurais/metabolismo , Vias Neurais/patologia , Vias Neurais/fisiopatologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/terapia , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/metabolismo , Neurônios/patologia , Estresse Oxidativo/genética , Fenilalanina/metabolismo , Fenilcetonúrias/metabolismo , Fenilcetonúrias/terapia , Substância Negra/metabolismo , Substância Negra/fisiopatologia , Resultado do TratamentoRESUMO
Tetrahydrobiopterin (BH(4)) is an essential cofactor for several enzymes, including all three forms of nitric oxide synthases, the three aromatic hydroxylases, and glyceryl-ether mono-oxygenase. A proper level of BH(4) is, therefore, necessary for the metabolism of phenylalanine and the production of nitric oxide, catecholamines, and serotonin. BH(4) deficiency has been shown to be closely associated with diverse neurological psychiatric disorders. Sepiapterin reductase (SPR) is an enzyme that catalyzes the final step of BH(4) biosynthesis. Whereas the number of cases of neuropsychological disorders resulting from deficiencies of other catalytic enzymes involved in BH(4) biosynthesis and metabolism has been increasing, only a handful of cases of SPR deficiency have been reported, and the role of SPR in BH(4) biosynthesis in vivo has been poorly understood. Here, we report that mice deficient in the Spr gene (Spr(-/-)) display disturbed pterin profiles and greatly diminished levels of dopamine, norepinephrine, and serotonin, indicating that SPR is essential for homeostasis of BH(4) and for the normal functions of BH(4)-dependent enzymes. The Spr(-/-) mice exhibit phenylketonuria, dwarfism, and impaired body movement. Oral supplementation of BH(4) and neurotransmitter precursors completely rescued dwarfism and phenylalanine metabolism. The biochemical and behavioral characteristics of Spr(-/-) mice share striking similarities with the symptoms observed in SPR-deficient patients. This Spr mutant strain of mice will be an invaluable resource to elucidate many important issues regarding SPR and BH(4) deficiencies.
Assuntos
Oxirredutases do Álcool/genética , Modelos Animais de Doenças , Erros Inatos do Metabolismo/genética , Oxirredutases do Álcool/deficiência , Animais , Sequência de Bases , Biopterinas/análogos & derivados , Biopterinas/biossíntese , Catecolaminas/biossíntese , Primers do DNA , Crescimento , Humanos , Imuno-Histoquímica , Locomoção , Camundongos , Camundongos Knockout , Fenótipo , Fenilalanina/metabolismo , Serotonina/biossínteseRESUMO
The Pah(enu2) mouse, created through ethylnitrosurea mutagenesis, is a model for phenylketonuria. These mice have elevated serum phenylalanine levels, hypopigmentation, and behavior and movement abnormalities, and female mice exhibit a maternal phenylketonuria syndrome. We evaluated the brains of adult and juvenile Pah(enu2) mice for consistent, demonstrable lesions to elucidate various neuropathologic processes and to assess the efficacy of various treatment modalities such as AAV-mediated gene therapy. One aspect of the disease may involve the effect of hyperphenylalanemia on catecholamine function. High levels of phenylalanine inhibit enzymes that are important in the conversion of tyrosine and tryptophan to their respective neurotransmitter derivatives, including dopamine. Therefore, assessment of dopaminergic regions was of interest in this study. Histologic evaluation of juvenile and adult brains revealed an increased cellular density as early as 4 wk of age in the middle to posterior hypothalamus and substantia nigra. The infiltrating cells showed immunoreactivity for CD11b and had morphologic characteristics of macrophages. There was marked expression of inducible nitric oxide synthase in these dopaminergic regions that co-localized to CD11b-positive cells. The CD11b-positive cells and increased inducible nitric oxide synthase expression in these regions may function in a neuroregulatory manner to compensate for alterations in dopamine metabolism.
Assuntos
Hipotálamo/patologia , Mesencéfalo/patologia , Fenilcetonúrias/genética , Animais , Antígeno CD11b/biossíntese , Catecolaminas/metabolismo , Modelos Animais de Doenças , Dopamina/metabolismo , Feminino , Técnicas de Transferência de Genes , Genótipo , Heterozigoto , Homozigoto , Hipotálamo/metabolismo , Imuno-Histoquímica , Macrófagos/metabolismo , Masculino , Mesencéfalo/metabolismo , Camundongos , Camundongos Mutantes , Neurônios/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Substância Negra/patologia , Fatores de Tempo , Triptofano/química , Tirosina/químicaRESUMO
The residue phenylalanine 198 (Phe 198) is a prominent cause of the lower activity of human carbonic anhydrase III (HCA III) compared with HCA II and other isozymes which have leucine at this site. We report the crystal structures of HCA III and the site-directed mutant F198L HCA III, both at 2.1 A resolution, and the enhancement of catalytic activity by exogenous proton donors containing imidazole rings. Both enzymes had a hexahistidine extension at the carboxy-terminal end, used to aid in purification, that was ordered in the crystal structures bound in the active site cavity of an adjacent symmetry-related enzyme. This observation allowed us to comment on a number of possible binding sites for imidazole and derivatives as exogenous proton donors/acceptors in catalysis by HCA III. Kinetic and structural evidence indicates that the phenyl side chain of Phe 198 in HCA III, about 5 A from the zinc, is a steric constriction in the active site, may cause altered interactions at the zinc-bound solvent, and is a binding site for the activation of catalysis by histidylhistidine. This suggests that sites of activation of the proton-transfer pathway in carbonic anhydrase are closer to the zinc than considered in previous studies.
Assuntos
Anidrase Carbônica III/química , Anidrase Carbônica III/metabolismo , Prótons , Catálise , Cristalografia por Raios X , Ativação Enzimática , Histidina/metabolismo , Humanos , Imidazóis/química , Cinética , Leucina/metabolismo , Lisina/metabolismo , Oligopeptídeos/metabolismo , Relação Estrutura-Atividade , Treonina/metabolismo , Zinco/químicaRESUMO
Expression constructs are subject to position-effects in transgenic assays unless they harbour elements that protect them from negative or positive influences exerted by chromatin at the site of integration. Locus control regions (LCRs) and boundary elements are able to protect from position effects by preventing heterochromatization of linked genes. The LCR in the human beta-globin gene locus is located far upstream of the genes and composed of several erythroid specific DNase I hypersensitive (HS) sites. Previous studies demonstrated that the LCR HS sites act synergistically to confer position-independent and high-level globin gene expression at different integration sites in transgenic mice. Here we show that LCR HS sites 2 and 3, in combination with boundary elements derived from the chicken beta-globin gene locus, confer high-level human beta-globin gene expression in different chromosomal integration sites in transgenic mice. Moreover, we found that the construct is accessible to nucleases and highly expressed when integrated in a centromeric region. These results demonstrate that the combination of enhancer, chromatin opening and boundary activities can establish independent expression units when integrated into chromatin.
Assuntos
Centrômero/genética , Cromatina/fisiologia , Globinas/genética , Região de Controle de Locus Gênico , Transgenes , Animais , Galinhas , Humanos , Camundongos , Camundongos TransgênicosRESUMO
The hydration of CO2 catalyzed by human carbonic anhydrase II (HCA II) is accompanied by proton transfer from the zinc-bound water of the enzyme to solution. We have replaced the proton shuttling residue His 64 with Ala and placed cysteine residues within the active-site cavity by mutating sites Trp 5, Asn 62, Ile 91, and Phe 131. These mutants were modified at the single inserted cysteine with imidazole analogs to introduce new potential shuttle groups. Catalysis by these modified mutants was determined by stopped-flow and 18O-exchange methods. Specificity in proton transfer was demonstrated; only modifications of the Cys 131-containing mutant showed enhancement in the proton transfer step of catalysis compared with unmodified Cys 131-containing mutant. Modifications at other sites resulted in up to 3-fold enhancement in rates of CO2 hydration, with apparent second-order rate constants near 350 microM(-1) s(-1). These are among the largest values of kcat/Km observed for a carbonic anhydrase.
Assuntos
Anidrase Carbônica II/metabolismo , Histidina/análogos & derivados , Sítios de Ligação , Dióxido de Carbono/metabolismo , Anidrase Carbônica II/genética , Humanos , Concentração de Íons de Hidrogênio , Cinética , Mutação , Estrutura Terciária de Proteína , PrótonsRESUMO
Glucose deprivation dramatically increases glucose transport activity in 3T3-L1 adipocytes without changing the concentration of GLUT1 in the plasma membrane (PM). Recent data suggest that subcompartments within the PM, specifically lipid rafts, may sequester selected proteins and alter their activity. To evaluate this possibility, we examined the distribution of GLUT1 in Triton X-100-soluble and -insoluble fractions. Our data show that 77% of the GLUT1 pool in PMs isolated from control 3T3-L1 adipocytes was extracted by 0.2% Triton X-100. After glucose deprivation for 12 h, only 56% of GLUT1 was extracted by detergent. In contrast, there was a twofold increase in the GLUT1 content of the detergent-resistant fraction. To evaluate whether GLUT1 interacts with a specific protein within lipid rafts, we focused on stomatin, recently shown to interact with and inhibit GLUT1 activity. Stomatin is distributed about equally between the PM and the biosynthetic compartments, and its expression is not affected by glucose deprivation. Nearly 90% of the PM pool of stomatin is in detergent-resistant lipid rafts. In normal 3T3-L1 adipocytes, we were unable to demonstrate an interaction between GLUT1 and stomatin in coimmunoprecipitation experiments. However, in stomatin-overexpressing cells, there was clear coprecipitation of stomatin with GLUT1 antibodies. Glucose deprivation increased this interaction threefold, which may reflect the increase of GLUT1 in lipid rafts. Despite this, there was little change in transport activity in glucose-deprived, stomatin-overexpressing cells vs. that in control cells. Thus GLUT1 interacts with stomatin in lipid rafts, but this interaction per se does not alter transport activity. Rather, stomatin may serve as an anchor for GLUT1 in lipid rafts, the environment of which favors activation.
Assuntos
Adipócitos/metabolismo , Glucose/fisiologia , Microdomínios da Membrana/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Células 3T3 , Animais , Proteínas Sanguíneas/antagonistas & inibidores , Proteínas Sanguíneas/imunologia , Northern Blotting , Western Blotting , Linhagem Celular , Transportador de Glucose Tipo 1 , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/imunologia , Camundongos , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismoRESUMO
Using laser desorption-atmospheric pressure chemical ionization we describe a novel approach for coupling mass spectrometry to polyacrylamide gel electrophoresis. In contrast to other approaches, the method allows for the direct sampling of a polyacrylamide gel-embedded protein without the addition of any exogenous matrixes and is performed at atmospheric pressure. After electrophoresis and enzymatic digestion, the gel is analyzed at AP by photons that desorb neutral peptide molecules, followed by corona discharge ionization in the gas-phase, and subsequent mass analysis. Our experimental results demonstrate the method to (1) rapidly identify electrophoresed proteins via "peptide fingerprinting" using protein databases, (2) detect single-amino acid polymorphisms, and (3) has potential for sub-picomole sensitivity while still maintaining in situ gel desorption-ionization at ambient conditions.
Assuntos
Pressão Atmosférica , Eletroforese em Gel de Poliacrilamida/métodos , Espectrometria de Massas/métodos , Análise de Sequência de Proteína/métodos , Anidrases Carbônicas/análise , Anidrases Carbônicas/genética , Bases de Dados de Proteínas , Humanos , Mioglobina/análise , Mutação Puntual , Análise de Sequência de Proteína/instrumentaçãoRESUMO
Functional constraints on proteins limit their evolutionary rates at specific sites. These constraints allow for the interpretation of conserved residues and sites with a rate change as those most likely underlying the functional similarities and differences among protein subfamilies, respectively. This study describes new likelihood-ratio tests (LRTs) that complement existing ones for the identification of both conserved and rate change sites. These identifications are validated by the recovery of residues that are known from existing biochemical and structural information to be critical for the functional similarities and differences among carbonic anhydrases (CAs). In combination with this other information, these LRTs also support a unique antioxidant defense role for the puzzling CA III. As illustrated by the CAs, these LRTs, in combination with other biological evidence, offer a powerful and cost-effective approach for testing hypotheses, making predictions, and designing experiments in protein functional studies.
Assuntos
Anidrases Carbônicas/genética , Sequência Conservada/genética , Evolução Molecular , Proteínas/genética , Sequência de Aminoácidos/genética , Animais , Antioxidantes/metabolismo , Anidrases Carbônicas/química , Simulação por Computador , Humanos , Cinética , Funções Verossimilhança , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Proteínas/metabolismo , Reprodutibilidade dos Testes , Projetos de Pesquisa , Homologia de Sequência de AminoácidosRESUMO
The maximal turnover rate of CO2 hydration catalyzed by the carbonic anhydrases is limited by proton transfer steps from the zinc-bound water to solution, steps that regenerate the catalytically active zinc-bound hydroxide. Catalysis of CO2 hydration by wild-type human carbonic anhydrase III (HCA III) (k(cat) = 2 ms (-1)) is the least efficient among the carbonic anhydrases in its class, in part because it lacks an efficient proton shuttle residue. We have used site-directed mutagenesis to test positions within the active-site cavity of HCA III for their ability to carry out proton transfer by replacing various residues with histidine. Catalysis by wild-type HCA III and these six variants was determined from the initial velocity of hydration of CO2 measured by stopped-flow spectrophotometry and from the exchange of 18O between CO2 and H2O at chemical equilibrium by mass spectrometry. The results show that histidine at three positions (Lys64His, Arg67His and Phe131His) have the capacity to transfer protons during catalysis, enhancing maximal velocity of CO2 hydration and 18O exchange from 4- to 15-fold compared with wild-type HCA III. Histidine residues at the other three positions (Trp5His, Tyr7His, Phe20His) showed no firm evidence for proton transfer. These results are discussed in terms of the stereochemistry of the active-site cavity and possible proton transfer pathways.
Assuntos
Anidrase Carbônica III/química , Prótons , Sítios de Ligação , Anidrase Carbônica III/genética , Humanos , Concentração de Íons de Hidrogênio , Mutagênese Sítio-Dirigida , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genéticaRESUMO
Laser desorption-atmospheric pressure chemical ionization-mass spectrometry (LD-APCI-MS) is presented for the atmospheric pressure (AP) sampling of tryptic peptides directly from a polyacrylamide gel. In contrast to other gel sampling mass spectrometric approaches, this technique does not require the addition of any exogenous matrices to the gel to assist with ionization. In this arrangement, a CO(2) laser at 10.6 micro m is used to desorb intact neutral peptide molecules from the gel, followed by ionization in the gas-phase with APCI. The ions are then sampled via a heated capillary inlet and transferred to a quadrupole ion trap mass spectrometer for mass analysis. Preliminary results suggest the polyacrylamide gel electrophoresis-LD-APCI-MS technique provides several advantages that could translate into a more convenient, robust methodology for the rapid identification and characterization of proteins. Finally, strategies regarding the further development of the method are presented.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Espectrometria de Massas/métodos , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Animais , Pressão Atmosférica , Grupo dos Citocromos c/química , Grupo dos Citocromos c/metabolismo , Cavalos , Lasers , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Tripsina/metabolismoRESUMO
We have prepared a site-specific mutant of human carbonic anhydrase (HCA) II with histidine residues at positions 7 and 64 in the active site cavity. Using a different isozyme, we have placed histidine residues in HCA III at positions 64 and 67 and in another mutant at positions 64 and 7. Each of these histidine residues can act as a proton transfer group in catalysis when it is the only nonliganding histidine in the active site cavity, except His(7) in HCA III. Using an (18)O exchange method to measure rate constants for intramolecular proton transfer, we have found that inserting two histidine residues into the active site cavity of either isozyme II or III of carbonic anhydrase results in rates of proton transfer to the zinc-bound hydroxide that are antagonistic or suppressive with respect to the corresponding single mutants. The crystal structure of Y7H HCA II, which contains both His(7) and His(64) within the active site cavity, shows the conformation of the side chain of His(64) moved from its position in the wild type and hydrogen-bonded through an intervening water molecule with the side chain of His(7). This suggests a cause of decreased proton transfer in catalysis.
Assuntos
Anidrase Carbônica III/metabolismo , Anidrase Carbônica II/metabolismo , Prótons , Sítios de Ligação , Dióxido de Carbono/química , Anidrase Carbônica II/química , Anidrase Carbônica III/química , Histidina/química , Histidina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Isoenzimas/química , Isoenzimas/metabolismo , Mutagênese Sítio-Dirigida , Oxigênio/química , Oxigênio/metabolismo , Estrutura Terciária de Proteína , Água/química , Difração de Raios X , Zinco/químicaRESUMO
We have previously shown that glucose deprivation activates glucose transport in a time- and protein synthesis-dependent fashion in 3T3-L1 adipocytes, a mouse cell line. Coincident with this is loss of glycogen. Because glycogen phosphorylase (GP) is responsible for glycogen degradation, we have examined its regulation to determine the relationship between transport activation and glycogen turnover. We first cloned the adipose GP cDNA and found sequence similarity to rat and human liver GP. Because the mouse liver GP cDNA sequence was unavailable, we cloned this cDNA as well and showed 100% identity between mouse adipose and liver sequences. A 3.1 kb transcript was readily observed in total RNA isolated from mouse liver or adipose by Northern blot analysis but, surprisingly, not in either total or poly(A) selected RNA from 3T3-L1 adipocytes. To evaluate regulation in 3T3-L1 adipocytes, we amplified GP mRNA from total RNA using multiplex, semi-quantitative PCR but found that expression did not change in response to deprivation. GP protein levels did not change either. However, endogenous GP activity from glucose-deprived cells was significantly elevated relative to controls, due to an increase in the phosphorylated form of GP (GPa). Finally, we overexpressed GP to determine its direct influence on the glucose transport system. These results were negative, which suggests that the nutrient control of glucose transport and GP occurs independently despite kinetic similarities in transport activation and glycogen turnover.
Assuntos
Adipócitos/metabolismo , Glucose/deficiência , Glicogênio Fosforilase/metabolismo , Células 3T3 , Adipócitos/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico , Clonagem Molecular , DNA Complementar/biossíntese , DNA Complementar/química , Ativação Enzimática , Regulação da Expressão Gênica , Glucose/metabolismo , Transportador de Glucose Tipo 1 , Glicogênio Fosforilase/biossíntese , Glicogênio Fosforilase/genética , Isoenzimas/biossíntese , Fígado/enzimologia , Camundongos , Dados de Sequência Molecular , Proteínas de Transporte de Monossacarídeos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
The maximal velocity of catalysis of CO(2) hydration by human carbonic anhydrase II (HCA II) requires proton transfer from zinc-bound water to solution assisted by His 64. The catalytic activity of a site-specific mutant of HCA II in which His 64 is replaced with Ala (H64A HCA II) can be rescued by exogenous proton donors/acceptors, usually derivatives of imidazole and pyridine. X-ray crystallography has identified Trp 5 as a binding site of the rescue agent 4-methylimidazole (4-MI) on H64A HCA II. This binding site overlaps with the "out" position in which His 64 in wild-type HCA II points away from the zinc. Activation by 4-MI as proton donor/acceptor in catalysis was determined in the dehydration direction using (18)O exchange between CO(2) and water and in the hydration direction by stopped-flow spectrophotometry. Replacement of Trp 5 by Ala, Leu, or Phe in H64A HCA II had no significant effect on enhancement by 4-MI of maximal rate constants for proton transfer in catalysis to levels near 10(5) s(-1). This high activity for chemical rescue indicates that the binding site of 4-MI at Trp 5 in H64A HCA II appears to be a nonproductive binding site, although it is possible that a similarly effective pathway for proton transfer exists in the mutants lacking Trp 5. Moreover, the data suggest that the out position of His 64 considered alone is not active in proton transfer in HCA II. In contrast to isozyme II, the replacement of Trp 5 by Ala in HCA III abolished chemical rescue of k(cat) by imidazole but left k(cat)/K(m) for hydration unchanged. This demonstrates that Trp 5 contributes to the predominant productive binding site for imidazole, with a maximal level for the rate constant of proton transfer near 10(4) s(-1). This difference in the susceptibility of CA II and III to chemical rescue may be related to the more sterically constrained and electrostatically positive nature of the active site cavity of CA III compared with CA II. The possibility of nonproductive binding sites for exogenous proton donors offers an explanation for the unusually low value of the intrinsic kinetic barrier obtained by application of Marcus theory to chemical rescue of H64A HCA II.