RESUMEN
Using site-specific mutagenesis, we have constructed two mutants of Escherichia coli dihydrofolate reductase (ecDHFR) to investigate further the function of a weakly acidic side chain at position 27 in substrate protonation: Asp27-->Glu (D27E) and Asp27-->Cys (D27C). The crystal structure of D27E ecDHFR in a binary complex with methotrexate shows that the side-chain oxygen atoms of Glu27 are in almost precisely the same location as those of Asp27 in the wild-type enzyme. Kinetic evidence indicates that Glu27 can indeed function efficiently in the proton relay to dihydrofolate. Even though vertebrate DHFRs all have a glutamic acid at the structurally equivalent position, the kinetic properties of Glu27 ecDHFR more closely resemble those of wild-type bacterial DHFRs than of vertebrate DHFRs. The D27C mutation produced an enzyme still capable of relaying a proton to dihydrofolate, but with the intrinsic pKa in its pH-activity profiles shifted upward to values characteristic of the more basic thiolate group. The crystal structure of the binary complex with methotrexate reveals two unexpected features: (1) the Cys27 sulfhydryl group does not point toward the pteridine-binding site, but the side chain of this residue is instead rotated 120 degrees to interact with a tyrosine side chain projecting from a neighboring beta-strand; (2) a bound ethanol molecule occupies a cavity adjacent to methotrexate. Ethanol is a component of the crystallization medium.
Asunto(s)
Tetrahidrofolato Deshidrogenasa/metabolismo , Asparagina/metabolismo , Sitios de Unión , Cristalografía , Escherichia coli/enzimología , Glutamatos/metabolismo , Ácido Glutámico , Humanos , Concentración de Iones de Hidrógeno , Cinética , Metotrexato/metabolismo , Mutagénesis Sitio-Dirigida , Protones , Pteridinas/metabolismo , Espectrofotometría Ultravioleta , Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolato Deshidrogenasa/genéticaRESUMEN
A remarkable correlation has been discovered between fluorescence lifetimes of bound NADPH and rates of hydride transfer among mutants of dihydrofolate reductase (DHFR) from Escherichia coli. Rates of hydride transfer from NADPH to dihydrofolate change by a factor of 1,000 for the series of mutant enzymes. Since binding constants for the initial complex between coenzyme and DHFR change by only a factor of 10, the major portion of the change in hydride transfer must be attributed to losses in transition-state stabilization. The time course of fluorescence decay for NADPH bound to DHFR is biphasic. Lifetimes ranging from 0.3 to 0.5 ns are attributed to a solvent-exposed dihydronicotinamide conformation of bound coenzyme which is presumably not active in catalysis, while decay times (tau 2) in the range of 1.3 to 2.3 ns are assigned to a more tightly bound species of NADPH in which dihydronicotinamide is sequestered from solvent. It is this slower component that is of interest. Ternary complexes with three different inhibitors, methotrexate, 5-deazafolate, and trimethoprim, were investigated, along with the holoenzyme complex; 3-acetylNADPH was also investigated. Fluorescence polarization decay, excitation polarization spectra, the temperature variation of fluorescence lifetimes, fluorescence amplitudes, and wavelength of absorbance maxima were measured. We suggest that dynamic quenching or internal conversion promotes decay of the excited state in NADPH-DHFR. When rates of hydride transfer are plotted against the fluorescence lifetime (tau 2) of tightly bound NADPH, an unusual correlation is observed. The fluorescence lifetime becomes longer as the rate of catalysis decreases for most mutants studied. However, the fluorescence lifetime is unchanged for those mutations that principally alter the binding of dihydrofolate while leaving most dihydronicotinamide interactions relatively undisturbed. The data are interpreted in terms of possible dynamic motions of a flexible loop region in DHFR which closes over both substrate and coenzyme binding sites. These motions could lead to faster rates of fluorescence decay in holoenzyme complexes and, when correlated over time, may be involved in other motions which give rise to enhanced rates of catalysis in DHFR.
Asunto(s)
Mutación , Tetrahidrofolato Deshidrogenasa/química , Catálisis , Coenzimas/química , Cinética , Sustancias Macromoleculares , Modelos Moleculares , NADP/química , Oxidación-Reducción , Unión Proteica , Soluciones , Espectrometría de Fluorescencia , Relación Estructura-Actividad , Temperatura , Tetrahidrofolato Deshidrogenasa/genéticaRESUMEN
We have applied site-directed mutagenesis methods to change the conserved tryptophan-22 in the substrate binding site of Escherichia coli dihydrofolate reductase to phenylalanine (W22F) and histidine (W22H). The crystal structure of the W22F mutant in a binary complex with the inhibitor methotrexate has been refined at 1.9-A resolution. The W22F difference Fourier map and least-squares refinement show that structural effects of the mutation are confined to the immediate vicinity of position 22 and include an unanticipated 0.4-A movement of the methionine-20 side chain. A conserved bound water-403, suspected to play a role in the protonation of substrate DHF, has not been displaced by the mutation despite the loss of a hydrogen bond with tryptophan-22. Steady-state kinetics, stopped-flow kinetics, and primary isotope effects indicate that both mutations increase the rate of product tetrahydrofolate release, the rate-limiting step in the case of the wild-type enzyme, while slowing the rate of hydride transfer to the point where it now becomes at least partially rate determining. Steady-state kinetics show that below pH 6.8, kcat is elevated by up to 5-fold in the W22F mutant as compared with the wild-type enzyme, although kcat/Km(dihydrofolate) is lower throughout the observed pH range. For the W22H mutant, both kcat and kcat/Km(dihydrofolate) are substantially lower than the corresponding wild-type values. While both mutations weaken dihydrofolate binding, cofactor NADPH binding is not significantly altered. Fitting of the kinetic pH profiles to a general protonation scheme suggests that the proton affinity of dihydrofolate may be enhanced upon binding to the enzyme. We suggest that the function of tryptophan-22 may be to properly position the side chain of methionine-20 with respect to N5 of the substrate dihydrofolate.
Asunto(s)
Escherichia coli/genética , Mutagénesis Sitio-Dirigida , Tetrahidrofolato Deshidrogenasa/fisiología , Triptófano/fisiología , Coenzimas/química , Deuterio , Escherichia coli/enzimología , Histidina/química , Histidina/genética , Histidina/fisiología , Concentración de Iones de Hidrógeno , Cinética , Metotrexato/química , NADP/metabolismo , Unión Proteica , Relación Estructura-Actividad , Especificidad por Sustrato , Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolato Deshidrogenasa/genética , Triptófano/química , Triptófano/genéticaRESUMEN
The reaction of ferric cytochrome c peroxidase (CcP) from Saccharomyces cerevisiae with peroxide produces compound I, characterized by both an oxyferryl iron center and a protein-based free radical. The electron paramagnetic resonance (EPR) signal of the CcP compound I radical can be resolved into a broad majority component which accounts for approximately 90% of the spin intensity and a narrow minority component which accounts for approximately 10% of the integrated spin intensity [Hori, H., & Yonetani, T. (1985) J. Biol. Chem. 260, 3549-3555]. It was shown previously that the broad component of the compound I radical signal is eliminated by mutation of Trp-191 to Phe [Scholes, C. P., Liu, Y., Fishel, L. F., Farnum, M. F., Mauro, J. M., & Kraut, J. (1989) Isr. J. Chem. 29, 85-92]. The present work probed the effect of mutations in the vicinity of this residue by EPR and electron-nuclear double resonance (ENDOR). These mutations were obtained from a plasmid-encoded form of S. cerevisiae expressed in Escherichia coli [Fishel, L. A., Villafranca, J. E., Mauro, J. M., & Kraut, J. (1987) Biochemistry 26, 351-360]. The EPR line shape and ENDOR signals of the compound I radical were perturbed only by mutations that alter Trp-191 or residues in its immediate vicinity: namely, Met-230 and Met-231, which have sulfur atoms within 4 A of the indole ring, and Asp-235, which forms a hydrogen bond with the indole nitrogen of Trp-191. Mutations of other potential oxidizable sites (tryptophan, tyrosine, methionine, and cysteine) did not alter the EPR line shapes of the compound I radical, although the integrated spin intensities were weaker in some of these mutants. Mutations at Met-230 and/or -231 perturbed the EPR line shapes of the compound I radical signal but did not eliminate it. ENDOR of these two methionine mutants showed alteration to the hyperfine couplings of several strongly coupled protons, which are characteristic of the majority compound I radical electronic structure, and a change in weaker hyperfine couplings, which suggests a different orientation of the radical with respect to its surroundings in the presence of these methionine mutations. Besides the Trp-191----Phe mutation, only the Asp-235----Asn mutation eliminated the broad component of the compound I signal. Loss of the broad compound I EPR signal coincides with both the loss of the Asp----Trp-191 hydrogen-bonding interaction and alteration of the position of the indole ring of Trp-191.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Citocromo-c Peroxidasa/metabolismo , Mutagénesis Sitio-Dirigida , Secuencia de Aminoácidos , Clonación Molecular , Codón/genética , Citocromo-c Peroxidasa/genética , Citocromo-c Peroxidasa/aislamiento & purificación , Espectroscopía de Resonancia por Spin del Electrón/métodos , Escherichia coli/genética , Radicales Libres , Espectroscopía de Resonancia Magnética/métodos , Metionina , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , TriptófanoRESUMEN
CO recombination to the cloned cytochrome c peroxidase [CCP(MI)] and mutants of CCP(MI) prepared by site-directed mutagenesis was examined as a function of pH by flash photolysis. The mutants examined included distal Arg 48----Leu, Lys; proximal Asp 235----Asn; and His 181----Gly. At alkaline pH, ferrous CCP(MI) was converted to a hexacoordinate form by a cooperative two-proton ionization, apparent pK(a) = 8.0. This change was observed in all of the mutants, although in the His 181----Gly mutant, the conversion to the hexacoordinate form was the result of a single-proton ionization, implicating His 181 as one of the two residues deprotonated in this isomerization. The pH-dependent conversion of CO ferrous CCP(MI) from acidic to alkaline forms was also observed and was similar to that reported for cytochrome c peroxidase from bakers' yeast [Iizuka, T., Makino, R., Ishimura, Y., & Yonetani, T. (1985) J. Biol. Chem. 260, 1407-1412]. Photolysis of the acidic form of the CO complex of CCP(MI) produces a kinetic form of the ferrous enzyme (form A) which exhibits the slow rate of CO recombination (l1' approximately 10(3) M-1 s-1) characteristic of peroxidases, while photolysis of the alkaline form of the CO complex produces a second kinetic form (form B), which exhibits a much faster rate of recombination (l2' approximately 10(5) M-1 s-1). Kinetic forms analogous to forms A and B were observed in all of the mutants examined. A third kinetic form (form B*) with a bimolecular rate constant l3' approximately 10(6) M-1 s-1 was also observed in the mutants at alkaline pH. Although the pH dependence for the conversion of form A to form B with increasing pH was altered by changes in the local heme environment, the rate of CO recombination by the respective forms was not dramatically altered in the mutants. Transient spectra of the reaction of CO with ferrous CCP(MI) after photolysis show that equilibrium between penta- and hexacoordinate ferrous enzyme is rapid relative to CO recombination. The presence of the internal sixth ligand has no discernible effect on the observed rate of recombination, however. The results presented indicate that in CCP(MI) the rate of ligand binding is determined primarily by isomerization of the protein from a closed conformation at acidic pH to an open conformation at alkaline pH and that polar effects of proximal Asp 235 and distal Arg 48 are of minor significance in the rate of CO recombination in both conformations.
Asunto(s)
Monóxido de Carbono/metabolismo , Citocromo-c Peroxidasa/metabolismo , Hemo/metabolismo , Mutación , Peroxidasas/metabolismo , Sitios de Unión , Clonación Molecular , Citocromo-c Peroxidasa/genética , Escherichia coli/genética , Genes , Concentración de Iones de Hidrógeno , Cinética , Matemática , Modelos Teóricos , Unión Proteica , Proteínas Recombinantes/metabolismo , EspectrofotometríaRESUMEN
Bovine plasma amine oxidase (PAO) has previously been shown to catalyze a nonstereospecific loss of tritium from [2(R)-3H]- and [2(S)-3H]dopamines, attributed to multiple, catalytically active binding sites for substrate [Summers, M. C., Markovic, R., & Klinman, J. P. (1979) Biochemistry 18, 1969-1979]. Analysis of products formed from incubation of dopamine with PAO in tritiated water indicates a stereospecific, pro-R, incorporation of label at C-2. Thus, tritium washout (random) and washin (pro-R) are not the microscopic reverse of one another. We conclude that the (enamine) intermediates leading to tritium washin are nonequivalently bound. The observation of pro-R incorporation has provided a straightforward synthetic route to [1(R)-2H,2(R)-3H]- and [1(S)-2H,2(R)-3H]dopamines, which upon oxidation with PAO are expected to be processed preferentially by 1S and 1R cleavage, respectively. From previously measured isotope effects, we predict the loss of tritium from the 1(R)-2H and 1(S)-2H samples to be 74:8 for a syn relationship between cleavage at C-1 and C-2 vs. 21:90 for an anti relationship. The observation of a 68:18 ratio at 100% conversion provides strong evidence for a syn cleavage. The data support a mechanism in which a single base catalyzes a 1,3-prototrophic shift of hydrogen from C-1 of the substrate to cofactor, followed by exchange from C-2. Additionally, the results confirm the presence of alternate binding modes for dopamine at the active site of bovine plasma amine oxidase. This interaction of dopamine with plasma amine oxidase is a rare example of mirror-image catalysis in which a single substrate has two functional binding orientations on an enzyme surface.