RESUMEN
The aim of this study was to investigate the role of mitochondrial ionic homeostasis in promoting reoxygenation-induced hypercontracture in cardiac muscle. Mitochondrial membrane potential and intramitochondrial Ca2+ concentration ([Ca2+]) were measured using confocal imaging in guinea pig ventricular myocytes exposed to anoxia and reoxygenation. Anoxia produced a variable, but often profound, mitochondrial depolarization. Some cells mounted a recovery of their mitochondrial membrane potential during reoxygenation; the depolarization was sustained in other cells. Recovery of the mitochondrial membrane potential seemed essential to avoid reoxygenation-induced hypercontracture. Reoxygenation also caused a sizable elevation in intramitochondrial [Ca2+], the amplitude of which was correlated with the likelihood of a cell undergoing hypercontracture. A sustained Ca2+ load analogous to that seen during reoxygenation was imposed on cardiac mitochondria through permeabilization of the plasma membrane. Elevation of intracellular [Ca2+] to 800 nM caused a substantial mitochondrial depolarization. We propose that the conditions seen in guinea pig ventricular myocytes during reoxygenation are well suited to produce Ca2+-dependent mitochondrial depolarization, which may play a significant role in promoting irreversible cell injury.
Asunto(s)
Calcio/metabolismo , Hipoxia de la Célula , Corazón/fisiología , Membranas Intracelulares/fisiología , Mitocondrias Cardíacas/fisiología , Animales , Permeabilidad de la Membrana Celular , Núcleo Celular/fisiología , Células Cultivadas , Cobayas , Cinética , Potenciales de la Membrana , Microscopía Confocal , Membrana Nuclear/fisiología , Oxígeno , Factores de TiempoRESUMEN
The aim of this study was to evaluate whether the magnitude and time course of the intracellular acidification observed in anoxic cardiac myocytes was sufficient to protect against reoxygenation-induced hypercontracture. Cytosolic [Ca2+], [Na+], and pH were measured using fluorescent indicators in myocytes that were first subjected to both anoxia and glucose deprivation and then oxygen and glucose restoration 15-30 min after the onset of rigor. The cytosol underwent a profound acidification early in anoxia (pH 7.21 to 6.84) that reached a plateau at the time of rigor contracture. In contrast, [Na+] rose throughout anoxia. Cytosolic [Ca2+] underwent little rise during anoxia, but reoxygenation induced a large spike in [Ca2+]. Reoxygenation also induced a significant secondary acidification of the cytosol that was apparently induced by the spike in [Ca2+]. The characteristics of this secondary acidification were deemed sufficient to provide partial protection against the hypercontracture associated with the reoxygenation-induced [Ca2+] transient.
Asunto(s)
Calcio/metabolismo , Hidrógeno/metabolismo , Hipoxia/metabolismo , Miocardio/metabolismo , Oxígeno/farmacología , Sodio/metabolismo , Animales , Citosol/metabolismo , Cobayas , Concentración de Iones de Hidrógeno , Miocardio/patología , Concentración OsmolarRESUMEN
Various photoactive phenothiazines were synthesized that possessed a 2-azido, 3-azido, 2-benzoyl, or 1,3,4-trifluoro-2-azido functionality in combination with various modifications of the N-alkyl side chain. These phenothiazines were evaluated for their ability to inhibit the calmodulin-mediated activation of phosphodiesterase (PDE). All were active in inhibiting the action of calmodulin (CaM), but those possessing either a 3-azido and a 4-(4-methyl-1-piperazinyl)butyl side chain or a 2-benzoyl group and 3-(dimethylamino)propyl side chain proved to be most active (I50 = 14 +/- 3 microM and 7 +/- 1 microM, respectively) when compared to the known inhibitor, chlorpromazine (CPZ, I50 = 30 microM). Calmodulin was photolabeled with ca. 35% efficiency in a light- and calcium-dependent fashion using a radiolabeled analog, 3-azido-10-(4-(4-[14C]methyl-1-piperazinyl)butyl)phenothiazine, of one of these compounds. Competition studies using this radiolabeled analog and CPZ were consistent with binding to one or both of the hydrophobic binding pockets of CaM.
Asunto(s)
Calmodulina/química , Fenotiazinas/síntesis química , Marcadores de Afinidad/síntesis química , Marcadores de Afinidad/química , Animales , Sitios de Unión , Unión Competitiva , Bovinos , Reactivos de Enlaces Cruzados , Activación Enzimática , Técnicas In Vitro , Estructura Molecular , Fenotiazinas/química , Fenotiazinas/efectos de la radiación , Hidrolasas Diéster Fosfóricas/metabolismo , FotoquímicaRESUMEN
The development of targeted, bidentate photoaffinity reagents for mapping the interacting domains of calmodulin (CaM) with the enzymes that it regulates required the synthesis and evaluation of the binding affinity of various phenothiazines. These photoaffinity reagents would possess a photoactive 3-azidophenothiazine group for cross-linking the hydrophobic binding domain of CaM, a second photoactive benzophenone group that would be activated at a different wavelength than the 3-azidophenothiazine group, and a suitable radiolabel. Difficulties were encountered in identifying those structural features that would be compatible with the introduction of a benzophenone group, with the solubility of these benzophenone-substituted phenothiazines, and with the ability of these phenothiazines to inhibit the calmodulin-mediated activation of phosphodiesterase. Solutions to this problem involved the preparation of phenothiazines possessing a quaternary ammonium salt, a zwitterionic amino acid, or a carbohydrate moiety. The phenothiazines that possessed photoactive 3-azido and benzophenone groups and in which one of the piperazine nitrogens in the side chain was converted to a quaternary, N-methylammonium iodide inhibited the calmodulin-mediated activation of phosphodiesterase at a level comparable to that of chlorpromazine.
Asunto(s)
Marcadores de Afinidad/síntesis química , Calmodulina/química , Fenotiazinas/síntesis química , Marcadores de Afinidad/química , Animales , Sitios de Unión , Calmodulina/metabolismo , Bovinos , Técnicas In Vitro , Estructura Molecular , Fenotiazinas/química , Hidrolasas Diéster Fosfóricas/metabolismo , FotoquímicaRESUMEN
Previous NMR studies on the ternary complex of human dihydrofolate reductase (hDHFR) with methotrexate (MTX) and NADPH detected six long-lived bound water molecules. Two of the water molecules, WatA and WatB, stabilize the structure of the protein while the other four, WatC, WatD, WatE and WatF, are involved in substrate binding and specificity. WatE may also act as a proton shuttle during catalysis. Here, the contributions of individual residues to the binding of these water molecules are investigated by performing NMR experiments on ternary complexes of mutant enzymes, W24F, E30A and E30Q. W24 and E30 are conserved residues that form hydrogen bonds with WatE in crystal structures of DHFR. Nuclear Overhauser effects (NOEs) are detected between WatE and the protein in all the mutant complexes, hence WatE still has a long lifetime bound to the complex when one of its hydrogen-bonding partners is deleted or altered by mutagenesis. The NOEs for WatE are much weaker, however, in the mutants than in wild-type. The NOEs for the other water molecules in and near the active site, WatA, WatC, WatD and WatF, also tend to be weaker in the mutant complexes. Little or no change is apparent in the NOEs for WatB, which is located outside the active site, farthest from the mutated residues. The decreased NOE intensities for the bound water molecules could be caused by changes in the positions and/or lifetimes of the water molecules. Chemical shift and NOE data indicate that the mutants have structures very similar to that of wild-type hDHFR, with possible conformational changes occurring only near the mutated residues. Based on the lack of structural change in the protein and evidence for increased structural fluctuations in the active sites of the mutant enzymes, it is likely that the NOE changes are caused, at least in part, by decreases in the lifetimes of the bound water molecules.
Asunto(s)
Metotrexato/química , NADP/química , Tetrahidrofolato Deshidrogenasa/química , Agua/química , Secuencia de Aminoácidos , Ácido Glutámico/metabolismo , Humanos , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Relación Estructura-Actividad , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Triptófano/metabolismo , Agua/metabolismoRESUMEN
Dihydrofolate reductase is an intracellular target enzyme for folate antagonists, including the anticancer drug methotrexate. In order to design novel drugs with altered binding properties, a detailed description of protein-drug interactions in solution is desirable to understand the specificity of drug binding. As a first step in this process, heteronuclear three-dimensional NMR spectroscopy has been used to make sequential resonance assignments for more than 90% of the residues in human dihydrofolate reductase complexed with methotrexate. Uniform enrichment of the 21.5-kDa protein with 15N was required to obtain the resonance assignments via heteronuclear 3D NMR spectroscopy since homonuclear 2D spectra did not provide sufficient 1H resonance dispersion. Medium- and long-range NOE's have been used to characterize the secondary structure of the binary ligand-enzyme complex in solution.
Asunto(s)
Tetrahidrofolato Deshidrogenasa/química , Secuencia de Aminoácidos , Sitios de Unión , Ácido Fólico/metabolismo , Humanos , Hidrógeno , Espectroscopía de Resonancia Magnética/métodos , Metotrexato/química , Datos de Secuencia Molecular , Mutación , Isótopos de Nitrógeno , Conformación Proteica , Soluciones , Relación Estructura-Actividad , Tetrahidrofolato Deshidrogenasa/genéticaRESUMEN
Dihydrofolate reductase (DHFR) is an intracellular target enzyme for folate antagonist drugs, including methotrexate. In order to compare the binding of methotrexate to human DHFR in solution with that observed in the crystalline state, NMR spectroscopy has been used to determine the conformation of the drug bound to human DHFR in solution. In agreement with what has been observed in the crystalline state, NOE's identified protein and methotrexate protons indicate that methotrexate binds in a non-productive orientation. In contrast to what has been reported for E. coli DHFR in solution, only one bound conformation of methotrexate is observed.
Asunto(s)
Metotrexato/metabolismo , Tetrahidrofolato Deshidrogenasa/metabolismo , Sitios de Unión , Humanos , Espectroscopía de Resonancia Magnética/métodos , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Soluciones , Tetrahidrofolato Deshidrogenasa/químicaRESUMEN
Four spin-labeled inhibitors of dihydrofolate reductase (DHFR) have been synthesized, each of which has the 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) reporting group at a different distance from the 2,4-diaminopyrimidine moiety by which the inhibitors are anchored and oriented in the active site. Inhibitors in which the TEMPO group is attached by a short side chain are weakly bound to DHFR from bacteria (Streptococcus faecium and Lactobacillus casei), to the bovine enzyme and to recombinant human DHFR. However, binding is sufficiently tight, especially in the ternary complexes with NADPH, for recording of the EPR spectra of the bound ligands. The spectra indicate that when these inhibitors are bound to the enzyme the TEMPO group is highly immobilized with correlation time, tau c, 4-20ns. Inhibitors that have the reporter group attached to the glutamate moiety of methotrexate bind to all four DHFRs more tightly than the inhibitors with shorter side chains by factors of up to 10(6). However, in most complexes formed by the inhibitors with longer side chains immobilization of the TEMPO group is slight (tau c 0.2-4 ns). These results are in general agreement with predictions from X-ray crystallographic results including thermal factors but there are some unanticipated differences between some results for bacterial and eukaryotic enzymes. Three of the splin-labeled inhibitors would provide good probes for distance measurements in and around the active site of mammalian DHFR.
Asunto(s)
Óxidos N-Cíclicos/metabolismo , Antagonistas del Ácido Fólico , Tetrahidrofolato Deshidrogenasa/metabolismo , Animales , Bovinos , Óxidos N-Cíclicos/síntesis química , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Lacticaseibacillus casei/enzimología , Ligandos , Hígado/enzimología , NADP/metabolismo , Proteínas Recombinantes , Marcadores de Spin , Streptococcus/enzimología , Relación Estructura-ActividadRESUMEN
The active sites of all bacterial and vertebrate dihydrofolate reductases that have been examined have a tryptophan residue near the binding sites for NADPH and dihydrofolate. In cases where the three-dimensional structure has been determined by X-ray crystallography, this conserved tryptophan residue makes hydrophobic and van der Waals interactions with the nicotinamide moiety of bound NADPH, and its indole nitrogen interacts with the C4 oxygen of bound folate through a bridge provided by a bound water molecule. We have addressed the question of why even the very conservative replacement of this tryptophan by phenylalanine does not seem to occur naturally. Human dihydrofolate reductase with this replacement of tryptophan by phenylalanine has increased rate constants for dissociation of substrates and products and a considerably decreased rate of hydride transfer. These cause some changes in steady-state kinetic behavior, including substantial increases in Michaelis constants for NADPH and dihydrofolate, but there is also a 3-fold increase in kcat. The branched mechanistic pathway for this enzyme has been completely defined and is sufficiently different from that of wild-type enzyme to cause changes in some transient-state kinetics. The most critical changes resulting from the amino acid substitution appear to be a 50% decrease in stability and a decrease in efficiency from 69% to 21% under intracellular conditions.
Asunto(s)
Tetrahidrofolato Deshidrogenasa/metabolismo , Regulación Alostérica , Apoenzimas/metabolismo , Sitios de Unión , Humanos , Hidrógeno/química , Concentración de Iones de Hidrógeno , Cinética , Ligandos , Mutación , NADP/metabolismo , Fenilalanina/fisiología , Desnaturalización Proteica , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolatos/metabolismo , Triptófano/fisiologíaRESUMEN
The 2.3-A crystal structure of recombinant human dihydrofolate reductase (EC 1.5.1.3, DHFR) has been solved as a binary complex with folate (a poor substrate at neutral pH) and also as a binary complex with an inhibitor, 5-deazafolate. The inhibitor appears to be protonated at N8 on binding, whereas folate is not. Rotation of the peptide plane joining I7 and V8 from its position in the folate complex permits hydrogen bonding of 5-deazafolate's protonated N8 to the backbone carbonyl of I7, thus contributing to the enzyme's greater affinity for 5-deazafolate than for folate. In this respect it is likely that bound 5-deazafolate furnishes a model for 7,8-dihydrofolate binding and, in addition, resembles the transition state for folate reduction. A hypothetical transition-state model for folate reduction, generated by superposition of the DHFR binary complexes human.5-deazafolate and chicken liver.NADPH, reveals a 1-A overlap of the binding sites for folate's pteridine ring and the dihydronicotinamide ring of NADPH. It is proposed that this binding-site overlap accelerates the reduction of both folate and 7,8-dihydrofolate by simultaneously binding substrate and cofactor with a sub van der Waals separation that is optimal for hydride transfer.
Asunto(s)
Tetrahidrofolato Deshidrogenasa/química , Sitios de Unión , Ácido Fólico/análogos & derivados , Ácido Fólico/química , Ácido Fólico/farmacología , Antagonistas del Ácido Fólico , Humanos , Metotrexato/química , Modelos Moleculares , Estructura Molecular , NADP/química , Conformación Proteica , Difracción de Rayos XRESUMEN
The role of the active site residue phenylalanine-31 (Phe31) for recombinant human dihydrofolate reductase (rHDHFR) has been probed by comparing the kinetic behavior of wild-type enzyme (wt) with mutant in which Phe31 is replaced by leucine (F31L rHDHFR). At pH 7.65 the steady-state kcat is almost doubled, but the rate constant for hydride transfer is decreased to less than half that for wt enzyme, as is the rate of the obligatory isomerization of the substrate complex that precedes hydride transfer. Although steady-state measurements indicated that the mutation causes large increases in Km for both substrates, dissociation constants for many complexes are decreased. These apparent paradoxes are due to major mutation-induced decreases in rate constants (koff) for dissociation of folate, dihydrofolate, and tetrahydrofolate from all of their complexes. This results in a mechanism proceeding almost entirely by only one of the two pathways used by wt enzyme. Other consequences of these changes are a much altered dependence of steady-state kcat on pH, inhibition rather than activation by tetrahydrofolate, absence of hysteresis in transient-state kinetics, and a decrease in enzyme efficiency under physiological conditions. The results indicate that there is no quantitative correlation between dihydrofolate binding and the rate of hydride transfer for this enzyme.
Asunto(s)
Proteínas Recombinantes/metabolismo , Tetrahidrofolato Deshidrogenasa/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Escherichia coli/enzimología , Humanos , Concentración de Iones de Hidrógeno , Cinética , Leucina , Fenilalanina/metabolismo , Tetrahidrofolatos/metabolismoRESUMEN
Association and dissociation rate constants obtained by stopped-flow spectroscopy have permitted definition of a kinetic scheme for recombinant human dihydrofolate reductase that correctly predicts full time course kinetics of the enzymatic reaction over a wide range of substrate and product concentrations. The scheme is complex compared with that for the bacterial enzyme and involves branched pathways. It successfully accounts for observed rapid hysteresis preceding steady state and for the nonhyperbolic dependence of steady-state rate on substrate and product concentrations. The major branch point in the catalytic cycle occurs at E.NADP.H4folate because either NADP or H4folate can dissociate from the ternary product complex (koff = 84 s-1 and 46 s-1, respectively). The rate of conversion of enzyme-bound substrates to products is very fast (k = 1360 s-1) and nearly unidirectional (Kequ = 37) so that other steps limit the catalytic rate. At saturating substrate concentrations these steps include release of NADP and H4folate from E.NADP.H4folate and release of products from the two abortive complexes E.NADPH.H4folate (koff = 225 s-1) and E.NADP.H4folate (koff = 4.6 s-1). Since NADP dissociates slowly from E.NADP.H2folate nearly 90% of the enzyme accumulates as this complex at steady state. Nonetheless, the catalytic rate is maintained at 12 s-1 by rapid flux of a small portion of the enzyme through an alternate branch. At physiological concentrations of substrates and products the steady-state rate is limited primarily by the rate of H2folate binding to E.NADPH so that the enzyme is extremely efficient.
Asunto(s)
Proteínas Recombinantes/metabolismo , Tetrahidrofolato Deshidrogenasa/metabolismo , Humanos , Cinética , Matemática , Modelos Teóricos , Unión Proteica , Espectrometría de Fluorescencia , Factores de TiempoRESUMEN
Transient and steady-state kinetics have been examined for dihydrofolate reductase (DHFR) from a number of sources. Rates of hydride transfer at pH 7.65 cover a wide range, from 7 s-1 for DHFR from a strain of Lactobacillus casei (LCDHFR1) to 3000 s-1 for recombinant human DHFR (rHDHFR). In all cases as the pH is increased from 7 to 10, Vmax for the steady-state reaction decreases, and DVmax, the primary isotope effect, increases. This indicates a decrease in the rate of hydride transfer with increasing pH. The cross-over points, at which rates of product release and hydride transfer become equal, were calculated to occur at DVmax = 2.34. The higher the rate of hydride transfer at pH 7.65, the higher the pH of the cross-over point. For LCDHFR1 the low rate of hydride transfer results in this process being partially rate-limiting for the steady-state reaction even at pH 5, with a cross-over point at about pH 7. At pH 7.65 the burst phase associated with the initial conversion of enzyme-bound substrates to enzyme-bound products has an isotope effect of 3 or higher for LCDHFR and for DHFR from Escherichia coli (ECDHFR). In contrast, the vertebrate DHFRs (bovine, BDHFR; chicken, CDHFR; and rHDHFR) exhibit a burst of product formation which is only partially limited by hydride transfer at this pH (Dkb: 2.3, 2.2, and 2.1, respectively). An obligatory isomerization of the ternary substrate complex or of the ternary product complex is postulated to be partially rate-limiting for the vertebrate enzymes. At pH 5 LCDHFR1 and ECDHFR also exhibit evidence of such a rate-limiting obligatory conformational transition of the substrate or product ternary complex.
Asunto(s)
Transporte de Electrón , Escherichia coli/enzimología , Lacticaseibacillus casei/enzimología , Tetrahidrofolato Deshidrogenasa/metabolismo , Animales , Sitios de Unión , Catálisis , Bovinos , Pollos , Deuterio , Humanos , Concentración de Iones de Hidrógeno , Cinética , Ratones , NADP/metabolismoRESUMEN
Oligonucleotide-directed, site-specific mutagenesis was used to convert phenylalanine-31 of human recombinant dihydrofolate reductase (DHFR) to leucine. This substitution was of interest in view of earlier chemical modification studies (Kumar et al., 1981) and structural studies based on X-ray crystallographic data (Matthews et al., 1985a,b) which had implicated the corresponding residue in chicken liver DHFR, Tyr-31, in the binding of dihydrofolate. Furthermore, this particular substitution allowed testing of the significance of protein sequence differences between mammalian and bacterial reductases at this position with regard to the species selectivity of trimethoprim. Both wild-type (WT) and mutant (F31L) enzymes were expressed and purified by using a heterologous expression system previously described (Prendergast et al., 1988). Values of the inhibition constants (Ki values) for trimethoprim were 1.00 and 1.08 microM for WT and F31L, respectively. Thus, the presence of phenylalanine at position 31 in human dihydrofolate reductase does not contribute to the species selectivity of trimethoprim. The Km values for nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) and dihydrofolate were elevated 10.8-fold and 9.4-fold, respectively, for the mutant enzyme, whereas the Vmax increased only 1.8-fold. Equilibrium dissociation constants (KD values) were obtained for the binding of NADPH and dihydrofolate in binary complexes with each enzyme. The KD for NADPH is similar in both WT and F31L, whereas the KD for dihydrofolate is 43-fold lower in F31L. Values for dihydrofolate association rate constants (kon) with enzyme and enzyme-NADPH complexes were measured by stopped-flow techniques.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Leucina/metabolismo , Fenilalanina/metabolismo , Tetrahidrofolato Deshidrogenasa/metabolismo , Sitios de Unión , Ácido Fólico/análogos & derivados , Ácido Fólico/farmacología , Humanos , Cinética , Leucina/fisiología , Metotrexato/farmacología , Mutación , NADP/metabolismo , Fenilalanina/fisiología , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Tetrahidrofolato Deshidrogenasa/genética , Trimetoprim/farmacología , Difracción de Rayos XRESUMEN
The transient state kinetics of catalysis for dihydrofolate reductase (DHFR) from several enzyme sources including highly purified recombinant human enzyme (rHDHFR) have been examined. Like DHFR from Escherichia coli, the enzyme from Lactobacillus casei, and isoenzyme 2 from Streptococcus faecium exhibit a slow increase in activity upon addition of substrates to enzyme. No slow hysteresis of this type was detected with recombinant human DHFR (rHDHFR) or DHFR from chicken or bovine liver or L1210 mouse leukemia cells (MDHFR). In contrast, both rHDHFR and MDHFR exhibited a very rapid decrease in activity (t1/2 = 30 and 20 ms, respectively) during a phase that occurred after the first turnover of the enzyme but before establishment of the steady state. This intermediate phase was not observed for the bacterial enzymes or the avian enzyme, nor was it observed with a mutant of rHDHFR in which Phe-31 has been replaced by leucine. For rHDHFR the intermediate phase is not a consequence of product inhibition, substrate depletion, or enzyme instability. It may therefore be concluded that this unusual transient state kinetic behavior results from the existence of two conformers of the enzyme, one of which has a higher turnover number than the other with the equilibrium shifting in favor of the less active conformer during the course of catalysis. The equilibrium is particularly favorable for the less active conformer when NADP is present in the active site of rHDHFR, whereas bound tetrahydrofolate favors the more active conformer. The more active conformer has a 6-fold higher Km for dihydrofolate than does the less active conformer. The existence of these conformers is likely to produce cooperative behavior by rHDHFR in vivo.
Asunto(s)
Proteínas Recombinantes/metabolismo , Tetrahidrofolato Deshidrogenasa/metabolismo , Animales , Pollos , Escherichia coli/enzimología , Humanos , Cinética , Lacticaseibacillus casei/enzimología , Leucemia L1210/enzimología , Ligandos , Hígado/enzimología , Matemática , Ratones , Especificidad de la Especie , Streptococcus/enzimología , Tetrahidrofolato Deshidrogenasa/genéticaRESUMEN
The binding site residue Trp-24 is conserved in all vertebrate and bacterial dihydrofolate reductases of known sequence. To determine its effects on enzyme properties, a Trp-24 to Phe-24 mutant (W-24-F) of human dihydrofolate reductase has been constructed by oligodeoxynucleotide site-directed mutagenesis. The W-24-F mutant enzyme appears to have a more open or flexible conformation as compared to the wild-type human dihydrofolate reductase on the basis of results of a number of studies. These studies include competitive ELISA using peptide-specific antibodies against human dihydrofolate reductase, thermal stability, and protease susceptibility studies of both mutant W-24-F and wild-type enzymes. It is concluded that Trp-24 is important for maintaining the structural integrity of the native enzymes. Changes in relative fluorescence quantum yield indicate that Trp-24 is buried and its fluorescence quenched relative to the other two tryptophan residues in the wild-type human reductase. Kinetic studies indicate that kcat values for W-24-F are increased in the pH range of 4.5-8.5 with a 5-fold increase at pH 7.5 as compared to the wild-type enzyme. However, the catalytic efficiency of W-24-F decreases rapidly as the pH is increased from 7.5 to 9.5. The Km values for dihydrofolate are also increased for W-24-F in the pH range of 4.5-9.5 with a 30-fold increase at pH 7.5, while the Km value for NADPH increases only ca. 1.4-fold at pH 7.5 as compared to the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Mutación , Fenilalanina , Tetrahidrofolato Deshidrogenasa/metabolismo , Triptófano , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , ADN/genética , Estabilidad de Enzimas , Ensayo de Inmunoadsorción Enzimática , Humanos , Cinética , Datos de Secuencia Molecular , Plásmidos , Espectrometría de Fluorescencia , Tetrahidrofolato Deshidrogenasa/genéticaRESUMEN
The kinetics of inhibitor binding to highly purified recombinant human dihydrofolate reductase (rHDHFR) have been examined. Methotrexate (MTX) binds rapidly (kon = 1.0 x 10(8) M-1 s-1) and tightly (koff/kon = 210 pM) to the preformed complex of rHDHFR with NADPH. The initial association reaction between rHDHFR.NADPH and MTX is followed by an isomerization of the resulting complex (kiso = 0.4 s-1) leading to a new conformer in which MTX is bound even more tightly (Ki = 3.4 pM). Similar results have been obtained with a major metabolite of MTX having four additional glutamate residues for which Ki = 1.4 pM. 7-HydroxyMTX, another major metabolite of MTX, is a weak inhibitor of rHDHFR (Ki = 8.9 nM), and a polyglutamate form of this metabolite is an equally weak inhibitor (Ki = 9.9 nM), so that the addition of glutamate residues to MTX or 7-hydroxyMTX has little effect on their binding. It follows that the significance of MTX polyglutamate formation relates to other roles such as increasing the cytotoxicity of MTX by prolonging intracellular retention of the drug. Another antifolate, trimethoprim, binds tightly to dihydrofolate reductases from bacterial sources, but weakly to rHDHFR in the ternary complex (KD = 0.5 microM). Although the association step is rapid (kon = 0.4 x 10(8) M-1 s-1), the dissociation rate is also rapid (koff = 15 s-1). Furthermore, there is no isomerization of the ternary complex of trimethoprim with rHDHFR, in contrast to the known isomerization of complexes of trimethoprim with bacterial dihydrofolate reductases.
Asunto(s)
Metotrexato/metabolismo , Proteínas Recombinantes/metabolismo , Tetrahidrofolato Deshidrogenasa/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Antagonistas del Ácido Fólico , Humanos , Isomerismo , Cinética , Metotrexato/farmacología , Unión Proteica , Proteínas Recombinantes/antagonistas & inhibidores , Espectrometría de Fluorescencia , Tetrahidrofolato Deshidrogenasa/genética , Trimetoprim/metabolismo , Trimetoprim/farmacologíaRESUMEN
A procaryotic high-level expression vector for human dihydrofolate reductase has been constructed and the protein characterized as a first step toward structure-function studies of this enzyme. A vector bearing the tac promoter, four synthetic oligodeoxynucleotides, and a restriction fragment from the dihydrofolate reductase cDNA were ligated in a manner which optimized the transcriptional and translational frequency of the enzyme mRNA. The reductase, comprising ca. 17% of the total soluble protein in the host bacteria, was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and characterized by amino acid composition, partial amino acid sequence, and steady-state kinetic analysis. This expression vector has been used as a template for double-stranded plasmid DNA site-specific mutagenesis. Functional studies on a Cys-6----Ser-6 mutant enzyme support the contention that Cys-6 is obligatory for organomercurial activation of human dihydrofolate reductase. The Ser-6 mutant enzyme was not activated to any extent following a 24-h incubation with p-(hydroxymercuri)benzoate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), whereas the kcat for Cys-6 reductase increased 2-fold under identical conditions. The specific activities of the Cys-6 and Ser-6 enzymes were virtually identical as determined by methotrexate titration as were the Km values for both dihydrofolate and NADPH. The Ser-6 mutant showed a decreased temperature stability and was more sensitive to inactivation by alpha-chymotrypsin when compared to the wild-type enzyme. These results suggest that the Ser-6 mutant reductase is conformationally altered relative to the Cys-6 native enzyme.
Asunto(s)
Mutación , Tetrahidrofolato Deshidrogenasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Escherichia coli/genética , Vectores Genéticos , Humanos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/síntesis química , Plásmidos , Tetrahidrofolato Deshidrogenasa/metabolismoRESUMEN
A series of 5,8-dideaza analogues of folic acid, isofolic acid, aminopterin, and isoaminopterin were evaluated for inhibition of thymidylate synthase, TS, from mouse L1210 leukemia cells with 10-propargyl-5,8-dideazafolic acid, CB3717, 4a, as the reference inhibitor. These compounds were also tested as inhibitors of human dihydrofolate reductase, DHFR, obtained from WIL2 cells. None of the analogues studied were as potent as 4a toward TS; however, 9-methyl-5,8-dideazaisoaminopterin, 6d, was only 2.5-fold less effective. Compound 4a was prepared by direct alkylation of the di-tert-butyl ester of 5,8-dideazafolic acid followed by hydrolysis of the resulting diethyl ester, which resulted from concomitant transesterification. It was found to be identical with a sample of 4a prepared by earlier methodology by using a variety of spectroscopic techniques. Its isomer, 9-propargyl-5,8-dideazaisofolic acid, 4b, which was synthesized by an analogous approach, was found to be dramatically less inhibitory toward TS than 4a. Each of the 2,4-diamino derivatives, including those possessing an allyl or propargyl group at N9, was an excellent inhibitor of DHFR, having a level of potency similar to that of methotrexate, MTX. However, many of these 5,8-dideazaaminopterin analogues were far more inhibitory toward TS than MTX.
Asunto(s)
Aminopterina/análogos & derivados , Antagonistas del Ácido Fólico , Ácido Fólico/análogos & derivados , Timidilato Sintasa/antagonistas & inhibidores , Aminopterina/farmacología , Animales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Ácido Fólico/farmacología , Humanos , Ratones , Relación Estructura-ActividadRESUMEN
A photoaffinity analogue of methotrexate, APA-[125I]ASA-Lys, specifically binds to dihydrofolate reductase and covalently modifies the enzyme following irradiation. An excess of methotrexate blocks incorporation of the photoprobe. Following cyanogen bromide digestion of the radiolabeled enzyme and high-pressure liquid chromatographic separation of the generated peptides, a majority of the label was centered around residues 63-65 (Lys-Asn-Arg), part of the inhibitor binding domain. This photoprobe is also transported into murine L1210 cells in a temperature-dependent, sulfhydryl reagent inhibitable manner with a Vmax similar to that for methotrexate. Ultraviolet irradiation at 4 degrees C of a cell suspension that had been incubated with the radiolabeled photoprobe resulted in the covalent modification of a 46-48 Kd protein. This can be demonstrated when the plasma membranes from the labeled cells are analyzed via sodium dodecylsulfate-polyacrylamide gel electrophoresis and autoradiography. Labeling of this protein occurs half-maximally at a reagent concentration that correlates with the Kt for transport of the iodinated compound. Protection against labeling of this protein by increasing amounts of methotrexate parallels the concentration dependence of inhibition of photoprobe uptake by methotrexate. In addition, no labeling occurs when a cell line that has a defective methotrexate transport system is similarly treated. Evidence that, in the absence of irradiation and at 37 degrees C, the iodinated probe is actually internalized is demonstrated by the labeling of two soluble proteins (Mr = 38 Kd and 21 Kd) derived from the cell homogenate supernatant.