RESUMEN
(-)-beta-D-2,6-Diaminopurine dioxolane (DAPD), is a nucleoside reverse transcriptase (RT) inhibitor with activity against human immunodeficiency virus type 1 (HIV-1). DAPD, which was designed as a water-soluble prodrug, is deaminated by adenosine deaminase to give (-)-beta-D-dioxolane guanine (DXG). By using calf adenosine deaminase a K(m) value of 15 +/- 0.7 microM was determined for DAPD, which was similar to the K(m) value for adenosine. However, the k(cat) for DAPD was 540-fold slower than the k(cat) for adenosine. In CEM cells and peripheral blood mononuclear cells exposed to DAPD or DXG, only the 5'-triphosphate of DXG (DXG-TP) was detected. DXG-TP is a potent alternative substrate inhibitor of HIV-1 RT. Rapid transient kinetic studies show the efficiency of incorporation for DXG-TP to be lower than that measured for the natural substrate, 2'-deoxyguanosine 5'-triphosphate. DXG-TP is a weak inhibitor of human DNA polymerases alpha and beta. Against the large subunit of human DNA polymerase gamma a K(i) value of 4.3 +/- 0.4 microM was determined for DXG-TP. DXG showed little or no cytotoxicity and no mitochondrial toxicity at the concentrations tested.
Asunto(s)
Fármacos Anti-VIH/farmacología , Dioxolanos/farmacología , Guanosina/análogos & derivados , VIH-1/efectos de los fármacos , Profármacos/farmacología , Nucleósidos de Purina/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Inhibidores de la Adenosina Desaminasa , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/microbiología , Células Cultivadas , ADN Viral/biosíntesis , Farmacorresistencia Microbiana , Inhibidores Enzimáticos/farmacología , Guanosina/farmacología , VIH-1/enzimología , VIH-1/ultraestructura , Humanos , Ácido Láctico/metabolismo , Microscopía Electrónica , Mitocondrias/efectos de los fármacos , Inhibidores de la Síntesis del Ácido NucleicoRESUMEN
The agouti protein plays an important role in the development of diabetes and obesity in rodents and has been shown to be a potent antagonist of melanocortin receptors. For this reason alanine-scanning mutagenesis was performed on the agouti protein carboxyl terminus to locate residues important for melanocortin receptor binding inhibition. When agouti residues Arg116 and Phe118 are changed to alanine, very large decreases in agouti affinity for melanocortin receptor 1, 3, and 4 result. Mutation of Phe117 to alanine causes a similar increase in agouti KI app at melanocortin receptor 4. Substitution of agouti residue Asp108 with alanine results in large increases in KI app for all three melanocortin receptors examined. All of these residues are conserved in the agouti-related transcript, ART, whose expression is up-regulated in animal models of obesity. The three-dimensional structure of the agouti carboxyl terminus was modeled, and residues which decrease receptor binding by a factor of > or = 15 when mutated to alanine localize to one side of the structure. These agouti variants with altered receptor selectivity may be useful in determining the role of melanocortin receptors in diabetes and obesity.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular , Proteínas/metabolismo , Receptores de Corticotropina/antagonistas & inhibidores , alfa-MSH/antagonistas & inhibidores , Proteína de Señalización Agouti , Secuencia de Aminoácidos , Animales , Sitios de Unión , Simulación por Computador , Análisis Mutacional de ADN , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Comunicación Paracrina , Unión Proteica , Proteínas/genética , Receptores de MelanocortinaRESUMEN
Several dominant mutations at the murine agouti locus cause a syndrome of marked obesity and insulin resistance. We have recently reported that intracellular free Ca2+ concentration ([Ca2+]i) is elevated in viable yellow mice. Because [Ca2+]i has a key role in the pathogenesis of insulin resistance, obesity, and hypertension, the role of the purified agouti gene product in regulating [Ca2+]i was evaluated in a number of cell types. Purified murine agouti induced slow, sustained increases in [Ca2+]i in A7r5 vascular smooth muscle cells and 3T3-L1 adipocytes in a dose-dependent fashion. In L6 skeletal myocytes, agouti stimulated an increase in [Ca2+]i with an apparent concentration eliciting 50% of the maximal response (EC50) of 62 nM. This response was substantially inhibited by Ca2+ entry blockade with nitrendipine. To determine whether melanocortin receptors play a role in agouti regulation of [Ca2+]i, we examined the effect of melanocortin peptides and agouti in cells stably transfected with human melanocortin receptors. Human embryonic kidney cells (HEK-293 cells) transfected with either the human melanocortin 1 receptor (MC1R) or melanocortin 3 receptor responded to human agouti with slow, sustained increases in [Ca2+]i, whereas nontransfected HEK-293 cells with no melanocortin receptors did not respond to agouti. Dose-response curves in the MC1R line showed that agouti had an EC50 of 18 nM, which is comparable to that for agouti antagonism of (125)I-Nle,D-Phe-alpha-melanocyte-stimulating hormone binding in the same cell line. This direct effect of agouti on stimulating increases in [Ca2+]i suggests a potential mechanism for agouti-induced insulin resistance.
Asunto(s)
Calcio/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Músculo Esquelético/metabolismo , Proteínas/farmacología , Receptor de Melanocortina Tipo 3 , Receptores de Corticotropina/fisiología , Adipocitos/metabolismo , Proteína de Señalización Agouti , Animales , Células Cultivadas , Citoplasma/metabolismo , Humanos , Hormonas Estimuladoras de los Melanocitos/farmacología , Ratones , Ratones Mutantes , Músculo Liso Vascular/metabolismo , Receptores de Melanocortina , Proteínas Recombinantes , TransfecciónRESUMEN
Several mutations that cause ectopic expression of the agouti gene result in obesity, hyperinsulinemia, and yellow coat color. A candidate pathway for agouti induced obesity and hyperinsulinemia is through altered signaling by melanocortin receptors, as agouti normally regulates coat coloration through antagonism of melanocortin receptor 1. Furthermore, melanocortin peptides mediate functions including steroidogenesis, lipolysis, and thermoregulation. We report apparent inhibition dissociation constants for mouse and human agouti protein inhibition of ligand binding to the melanocortin receptors, to determine which of these receptors might be involved in agouti induced diabetes. The similarity in the apparent K(I) values for agouti inhibition of ligand binding to the brain melanocortin receptors 3 and 4 (mouse: K(I) app = 190 +/- 74 and 54 +/- 18 nM; human: K(I) app = 140 +/- 56 and 70 +/- 18 nM, respectively) suggests that the MC3-R is a potential candidate for a receptor mediating the effects of agouti protein overexpression. Agouti residues important for melanocortin receptor inhibition were identified through the analysis of deletion constructs and site-specific variants. Val83 is important for inhibition of binding to MC1-R (K(I) app for Val83Ala agouti increased 13-fold relative to wild-type protein). Arg85, Pro86, and Pro89 are important for selective inhibition of binding between MC1-R and MC3-R and MC4-R as their apparent K(I) values are essentially unchanged at MC1-R, while they have increased 6-10-fold relative to wild-type protein at MC3-R and MC4-R.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular , Proteínas/farmacología , Receptor de Melanocortina Tipo 3 , Receptores de Corticotropina/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína de Señalización Agouti , Animales , Línea Celular , Humanos , Ligandos , Ratones , Mutagénesis Sitio-Dirigida , Mutación , Proteínas/genética , Proteínas/metabolismo , Receptores de MelanocortinaRESUMEN
Direct metal ligands to transition metals in metalloproteins exert a profound effect on protein-metal affinity and function. Indirect ligands, i.e., second-shell residues that hydrogen bond to direct metal ligands, typically exert more subtle effects on the chemical properties of the protein-metal complex. However, E117 of human carbonic anhydrase II (CAII), which is part of the E117-119-Zn(2+) triad, is a notable exception: E117-substituted CAIIs exhibit dramatically increased kinetics of zinc complexation, and the E117Q variant exhibits enormously diminished catalytic activity and sulfonamide affinity. The three-dimensional structures of zinc-bound and zinc-free E117Q CAII reveal no discrete structural changes in the active site that are responsible for enhanced zinc equilibration kinetics and decreased activity. Additionally, the structure of the acetazolamide complex is essentially identical to that of the wild-type enzyme despite the 10(4)-fold loss of enzyme-inhibitor affinity. We conclude, therefore, that the functional differences between E117Q and wild-type CAIIs arise from electrostatic and not structural differences in the active site. We propose that the E117Q substitution reverses the polarity of the residue 117-H119 hydrogen bond, thereby stabilizing H119 as a histidinate anion in the E117Q CAII holoenzyme. The additional negative charge in the first coordination sphere of the metal ion increases the pK(a) of the zinc-water ligand, destabilizes the transition state for CO(2) hydration, and facilitates the exchange of a zinc-histidine ligand with an additional water molecule by decreasing the stability of the tetrahedral zinc complex. These novel properties engineered into E117Q CAII facilitate the exploitation of CAII as a rapid and sensitive Zn(2+) biosensor.
Asunto(s)
Anhidrasas Carbónicas/química , Acetazolamida/química , Apoenzimas/química , Catálisis , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Cinética , Ligandos , Metaloproteínas , Mutagénesis Sitio-Dirigida , Mutación Puntual , Relación Estructura-Actividad , Zinc/químicaRESUMEN
The structure of histidine 94-->aspartate (H94D) carbonic anhydrase II (CAII) crystallized in an orthorhombic space group has been determined to 2.5 A resolution. This crystal form is not isomorphous with monoclinic wild-type enzyme crystals or with the monoclinic crystal form of H94D CAII reported earlier [Kiefer,L.L., Ippolito, J.A., Fierke, C.A. and Christianson, D.W. (1993) J. Am. Chem. Soc., 115, 12581-12582]. In monoclinic H94D CAII, a fully occupied zinc ion is tetrahedrally coordinated by D94, H96, H119 and a water molecule. In orthorhombic H94D CAII, a partially occupied zinc ion is coordinated by H96 and H119 and only weakly coordinated by a disordered D94 side chain. These differences are particularly surprising given that the two crystal forms co-precipitate in the same drop in the same experiment. Re-refinement of the orthorhombic crystal form of H94C CAII and comparison with its corresponding monoclinic crystal form yield similar results. It appears that partial-but not full-zinc dissociation accompanies the crystallization of CAII variants in the orthorhombic crystal form, and significant differences on the protein surface presumably affect the relative stability of each crystal lattice. These results underscore an unexpected ambiguity in this protein engineering experiment: which crystal structure of H94D CAII should be correlated with functional measurements made in solution?
Asunto(s)
Anhidrasas Carbónicas/química , Sitios de Unión , Anhidrasas Carbónicas/genética , Anhidrasas Carbónicas/metabolismo , Cristalización , Cristalografía por Rayos X , Electroquímica , Humanos , Técnicas In Vitro , Modelos Moleculares , Estructura Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , Conformación Proteica , Ingeniería de Proteínas , Zinc/metabolismoRESUMEN
Carbonic anhydrase II (CAII) contains a conserved His3 zinc polyhedron which is essential for catalysis. Removal of any one of the His ligands by replacement with Ala decreases (approximately 10(5)-fold), but does not abolish, zinc binding and increases the rate constant for zinc dissociation. CAII variants with a His ligand substituted with Cys, Asp, or Glu bind zinc only approximately 10-fold better than a His2 zinc polyhedron in CAII. The large decrease in zinc affinity (approximately 5 kcal/mol) in these variants compared to the wild-type His3 site reflects mainly unfavorable compensatory protein structural rearrangements observed in the X-ray crystallographic structures of some of these CAII variants, described by Ippolito and Christianson (following paper in this issue). However, the zinc affinity of these sites is still higher than zinc polyhedra designed de novo. Substitution of the His zinc ligands with negatively charged amino acids both increases the pKa of the zinc-bound water by > or = 1.6 pH units, confirming that neutral ligands maintain the low zinc-water pKa, and decreases the pH-independent kcat/KM for ester hydrolysis (3-30-fold) and CO2 hydration (approximately 10(3)-10(5)-fold). Additionally, decreases in the dissociation constant (approximately approximately 10(2)-10(5)-fold) for the transition state analog acetazolamide correlate with the decreased catalytic efficiency and increased pKa of these CAII variants. These data indicate that the histidine ligands, although not essential for catalysis, are conserved to maximize electrostatic stabilization of both the ground-state zinc-hydroxide and the negatively charged transition state. These studies provide valuable insights into the functional consequences of engineering a catalytic zinc site in a metalloenzyme.
Asunto(s)
Anhidrasas Carbónicas/química , Sitios de Unión , Dióxido de Carbono/química , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Cinética , Metaloproteínas/química , Mutagénesis Sitio-Dirigida , Solventes , Relación Estructura-Actividad , Agua/química , Zinc/químicaRESUMEN
Substitution of cysteine for threonine-199, the amino acid which hydrogen bonds with zinc-bound hydroxide in wild-type carbonic anhydrase II (CAII), leads to the formation of a new His3Cys zinc coordination polyhedron. The optical absorption spectrum of the Co(2+)-substituted threonine-199-->cysteine (T199C) variant and the three-dimensional structure [Ippolito, J. A., & Christianson, D. W. (1993) Biochemistry (following paper in this issue)] indicate that the new thiolate side chain coordinates to the metal ion, displacing the metal-bound solvent molecule. The engineered thiolate ligand increases zinc binding (4-fold) and decreases catalytic activity substantially (approximately 10(3)-fold) but not completely. However, this residual activity is due to an active species containing a zinc-bound solvent ligand with the cysteine-199 side chain occupying an alternate conformation. The equilibrium between these conformers reflects the energetic balance between the formation of the zinc-thiolate bond and structural rearrangements in the Ser-197-->Cys-206 loop necessary to achieve this metal coordination. This designed His3Cys metal polyhedron may mimic the zinc binding site in the matrix metalloproteinase prostromelysin.
Asunto(s)
Anhidrasas Carbónicas/química , Anhidrasas Carbónicas/metabolismo , Cisteína , Treonina , Zinc/metabolismo , Acetazolamida/farmacología , Secuencia de Bases , Sitios de Unión , Anhidrasas Carbónicas/genética , Cobalto/metabolismo , Humanos , Enlace de Hidrógeno , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Ligandos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Ingeniería de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrofotometría/métodosRESUMEN
The structure of the His-94-->Cys variant of human carbonic anhydrase II (CAII) has been determined by X-ray crystallographic methods to a resolution of 2.3 A with a final crystallographic R factor of 0.155. This variant of CAII crystallizes in orthorhombic space group P2(1)2(1)2(1) which is the first example of a new crystal form for this important zinc hydrase (the wild-type enzyme crystallizes in monoclinic space group P21 under similar crystallization conditions). Although the overall structure of the enzyme in the orthorhombic crystal form is similar to that of the wild-type protein in the monoclinic crystal form, the rms deviation of C alpha atoms between the two structures is 0.5 A. Larger structural deviations occur in regions of the protein molecule involved in crystal lattice contacts, and significant structural changes are found in the polypeptide strand containing Cys-94. Surprisingly, no electron density corresponding to a zinc ion is found in the active site of crystalline His-94-->Cys CAII, even though the stoichiometry of zinc binding to this variant in solution is confirmed by atomic absorption spectroscopy. However, the KD for zinc dissociation from the variant is increased 10(4)-fold compared with wild-type enzyme; furthermore, under the crystallization conditions of high ionic strength (1.75-2.5 M ammonium sulfate), the observed KD is increased further, which leads to zinc dissociation. Spectroscopic analysis of Co(2+)-substituted His-94-->Cys CAII indicates that the metal binds in a tetrahedral geometry with a new thiolate bond.(ABSTRACT TRUNCATED AT 250 WORDS)