Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:1.5.1.3 (dihydrofolate reductase)
5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using Escherichia coli guanosine triphosphate cyclohydrolase, dihydroneopterin triphosphate was synthesized from guanosine triphosphate and was compared with sepiapterin as a substrate for tetrahydrobiopterin formation in bovine adrenal medulla extracts. The dihydrofolate reductase inhibitor, methotrexate, blocks the formation of tetrahydrobiopterin from sepiapterin but not from dihydroneopterin triphosphate. Reduced nicotinamide adenine dinucleotide phosphate and a divalent metal ion are required in partially purified preparations (gel filtration of 40-60% ammonium sulfate fraction on Ultrogel ACA-34) for the biosynthesis of tetrahydrobiopterin from dihydroneopterin triphosphate. Sepiapterin was converted only to dihydrobiopterin in the same fractions since dihydrofolate reductase was removed. The evidence indicates that both dihydroneopterin triphosphate and sepiapterin are good precursors of tetrahydrobiopterin but they are not on the same pathway, contrary to previous proposals.
...
PMID:Tetrahydrobiopterin is synthesized by separate pathways from dihydroneopterin triphosphate and from sepiapterin in adrenal medulla preparations. 663 80

We have prepared a selectively deuterated dihydrofolate reductase in which all the aromatic protons except the C(2) protons of tryptophan have been replaced by deuterium and have examined the 1H NMR spectra of its complexes with folate, trimethoprim, methotrexate, NADP+, and NADPH. One of the four Trp C(2)-proton resonance signals (signal P at 3.66 ppm from dioxane) has been assigned to Trp-21 by examining the NMR spectrum of a selectively deuterated N-bromosuccinimide-modified dihydrofolate reductase. This signal is not perturbed by NADPH, indicating that the coenzyme is not binding close to the 2 position of Trp-21. This contrasts markedly with the 19F shift (2.7 ppm) observed for the 19F signal of Trp-21 in the NADPH complex with the 6-fluorotryptophan-labeled enzyme. In fact the crystal structure of the enzyme . methotrexate . NADPH shows that the carboxamide group of the reduced nicotinamide ring is near to the 6 position of Trp-21 but remote from its 2 position. The nonadditivity of the 1H chemical-shift contributions for signals tentatively assigned to Trp-5 and -133 indicates that these residues are influenced by ligand-induced conformational changes.
...
PMID:Proton nuclear magnetic resonance studies of the effects of ligand binding on tryptophan residues of selectively deuterated dihydrofolate reductase from Lactobacillus casei. 677 Aug 92

The binding, or association, constants of NADP+ NADPH, and a series of structural analogues to dihydrofolate reductase from Lactobacillus casei MTX/R have been determined fluorometrically. Modification of the adenine or nicotinamide rings has little effect on the binding of the oxidized coenzyme, but the thionicotinamide and acetylpyridine analogues of the reduced coenzyme bind much more weakly than NADPH itself. In the presence of the substrate, folate, or the inhibitors methotrxate or trimethoprim, the oxidized coenzymes bind appreciably more tightly to the enzyme. The magnitude of this "cooperativity", which covers a range of 1-37 fold, depends markedly on the structure of both the coenzyme and the substrate or substrate analogue; the nicotinamide ring of the coenzymes is clearly important in these effects. The binding constants of the reduced coenzymes in the presence of methotrexate or trimethoprim were too high to be measured fluorometrically. The dissociation rate constants of the coenzymes from their ternary complexes were therefore measured and compared with the values for the binary complexes reported by Dunn and co-workers [Dunn, S. M. J., Bathchelor, J. G., & King, R. W.(1978) Biochemistry 17, 2356]. The presence of the inhibitors leads to very substantial decreases in the coenzyme dissociation rate constant--by factors of 300-2200. The binding constant of methotrexate in the ternary complex is calculated to be approximately 1.3 X 10(12) M-1. The structural origins of the differences in binding constant and cooperative behavior of the various coenzymes and coenzyme analogues are discussed in the light of information from crystallography and NMR spectroscopy.
...
PMID:Binding of coenzyme analogues to Lactobacillus casei dihydrofolate reductase: binary and ternary complexes. 677 48

The chemical shifts of all the aromatic proton and anomeric proton resonances of NADP+, NADPH, and several structural analogues have been determined in their complexes with Lactobacillus casei dihydrofolate reductase by double-resonance (saturation transfer) experiments. The binding of NADP+ to the enzyme leads to large (0.9-1.6 ppm) downfield shifts of all the nicotinamide proton resonances and somewhat smaller upfield shifts of the adenine proton resonance. The latter signals show very similar chemical shifts in the binary and ternary complexes of NADP+ and the binary complexes of several other coenzymes, suggesting that the environment of the adenine ring is similar in all cases. In contrast, the nicotinamide proton resonances show much greater variability in position from one complex to another. The data show that the environments of the nicotinamide rings of NADP+, NADPH, and the thionicotinamide and acetylpyridine analogues of NADP+ in their binary complexes with the enzyme are quite markedly different from one another. Addition of folate or methotrexate to the binary complex has only modest effects on the nicotinamide ring of NADP+, but trimethoprim produces a substantial change in its environment. The dissociation rate constant of NADP+ from a number of complexes was also determined by saturation transfer.
...
PMID:Proton nuclear magnetic resonance saturation transfer studies of coenzyme binding to Lactobacillus casei dihydrofolate reductase. 677 50

The role of histidine residues of dihydrofolate reductase from Lactobacillus casei was investigated with diethyl pyrocarbonate. This enzyme has no cysteine residues and differs in this respect from many nicotinamide nucleotide dehydrogenases, which have catalytically important sulfhydryl groups. X-ray studies of this enzyme have shown that histidine residues are involved in substrate binding but not in proton transfer [Matthews et al. (1978) J. Biol. Chem. 253, 6946]. Dihydrofolate reductase was inactivated by diethyl pyrocarbonate; the second-order rate constant for the reaction was 29 M-1 min-1 at 0 degrees C. The difference spectrum of native and diethyl pyrocarbonate inactivated enzyme had a maximum near 242 nm, which indicated a reaction with histidine residues. The absence of any spectral difference near 280 nm indicated that diethyl pyrocarbonate had not reacted with tyrosine residues. Dihydrofolate reductase lost all of its enzymatic activity after about six of the seven histidine residues had been modified. No catalytic activity was lost during an initial rapid reaction with about four histidine residues, but a subsequent slower reaction involving an additional one or two residues was associated with the loss of activity. The enzyme was protected from inactivation by either of the substrates NADPH or dihydrofolate. In fact, treatment with diethyl pyrocarbonate in the presence of either substrate, but particularly with NADPH, resulted in substantially greater activity than that found with untreated enzyme. Treatment with 1 M hydroxylamine partially restored activity to dihydrofolate reductase that had been inactivated by diethyl pyrocarbonate.
...
PMID:Inactivation of dihydrofolate reductase from Lactobacillus casei by diethyl pyrocarbonate. 680 28

New details of NADPH binding to Lactobacillus casei dihydrofolate reductase have become visible as a result of crystallographic refinement to an R factor of 0.152 at 1.7 A resolution. Conformational torsion angles for bound NADPH have been extensively revised and specific interatomic contacts responsible for cofactor binding have been identified. In addition, several structurally conserved water molecules are seen to mediate the protein-ligand interaction. In the nicotinamide binding site three oxygen atoms of the enzyme lie in the plane of the pyridine ring and close to ring carbons 2, 4, and 6. The placement of these polar groups suggests that the enzyme stabilizes a C4-carbonium electronic isomer of oxidized nicotinamide in the transition state. Pyramidalization of ring nitrogen N1 in the transition state might be promoted by a fixed water molecule positioned to donate a hydrogen bond to the N1 lone pair orbital. Pyramidalization could also relieve an unfavorable steric contact due to the observed rotation of the nicotinamide's carboxamide group by 180 degrees from its most stable conformation.
...
PMID:Crystal structures of Escherichia coli and Lactobacillus casei dihydrofolate reductase refined at 1.7 A resolution. II. Environment of bound NADPH and implications for catalysis. 681 79

The Lactobacillus casei dihydrofolate reductase-folate-NADP+ complex is shown by 1H and 13C NMR to exist in three interconverting conformational states, I, IIa, and IIb. The proportions of the three states, as estimated from the intensities of the three separate 13C resonances observed in the complex containing [3-carboxamido-13C]NADP+, are pH dependent. State I predominates at low pH and states IIa and IIb predominate at high pH; the ratio IIa:IIb is pH independent. The pH dependence of the interconversion of states I and IIa + IIb can be explained by a model in which a group on the enzyme has a pK of less than 5 in state IIa + IIb and greater than 7 in state I. 1H, 13C, and 31P NMR has been used to characterize the structural differences between the three states of the complex. As judged by the 1H and 13C chemical shifts of the bound coenzyme, states I and IIa are similar to one another but quite different from state IIb. This difference appears to be a localized one, since only the nicotinamide 2 and 4 protons, nicotinamide 3-carboxamide 13C, and pteridine 7 proton show differences in chemical shift between these states. These differences are, however, large--up to 1.4 ppm for 1H and 2 ppm for 13C. The remaining coenzyme protons, as well as the three 31P nuclei, are unaffected. Studies of the C2 proton resonances of the seven histidine residues show that the ionizable group responsible for the interconversion of states I and IIa + IIb is not a histidine (although two histidines show slight differences in environment between states IIa and IIb); the possible identity of this ionizable group and the nature of the conformational differences between the states are discussed.
...
PMID:Hydrogen-1, carbon-13, and phosphorus-31 nuclear magnetic resonance studies of the dihydrofolate reductase-nicotinamide adenine dinucleotide phosphate-folate complex: characterization of three coexisting conformational states. 681 82

Reduced nicotinamide adenine dinucleotide phosphate (NADPH), folate, dihydrofolate, and the inhibitors trimethoprim and methotrexate bind rapidly and reversibly to both dihydrofolate reductase isoenzymes isolated from Escherichia coli RT500. The coenzyme and substrates appear to bind to only one of the mixture of two forms of the isoenzyme present at equilibrium, while the inhibitors bind to both forms. The proportions of the two forms are different for the two isoenzymes and are pH dependent in each case. The measured association rate constants for substrates and inhibitors lie in the range (1--2) x 10(-7) M-1 s-1 at 25 degrees C but are unlikely to be diffusion controlled. The rate constant for NADPH binding is 2 x 10(6) M-1 s-1. The formation of binary complexes takes place through a multistep mechanism. A minimum of three steps is required to explain the kinetic results. An equilibrium between two or more forms of the enzyme--ligand complex governs the overall dissociation. The stability of this equilibrium is largely responsible for the tighter binding of inhibitors relative to substrate or coenzyme and also for the different binding strengths of inhibitors to the isoenzymes.
...
PMID:Kinetics of substrate, coenzyme, and inhibitor binding to Escherichia coli dihydrofolate reductase. 701 78

The crystal and molecular structure of folic acid dihydrate has been determined by x-ray diffraction. Folic acid is in an extended conformation with the pteridine ring in the keto form. The C(4) oxygen and N(10) atoms are on the same side of the molecule, hydrogen-bonded to the same water. This conformation has the pteridine rotated approximately 180 degrees away from the orientation of the pteridine ring of methotrexate bound to dihydrofolate reductase. The folic acid pteridine and phenyl rings interact in a stacking manner which is suggestive of the type of associations these groups could form in a complex of folate, dihydrofolate reductase, and reduced nicotinamide adenine dinucleotide phosphate.
...
PMID:Folic acid: crystal structure and implications for enzyme binding. 742 95

Crystal structures of Escherichia coli dihydrofolate reductase (ecDHFR, EC 1.5.1.3) in binary complexes with folate, 5-deazafolate (5dfol), and 5,10-dideazatetrahydrofolate (ddTHF) have been refined to R-factors of 13.7%, 14.9%, and 14.5%, respectively, all at 1.9 A. All three are isomorphous with a previously reported binary complex of ecDHFR with methotrexate (MTX), in space group P6(1), two molecules per asymmetric unit [Bolin, J. T., Filman, D. J., Matthews, D. A., Hamlin, R. C., & Kraut, J. (1982) J. Biol. Chem. 257, 13650-13662]. A hitherto unobserved water molecule is hydrogen bonded to the pteridine N5 and O4 in both molecules of the asymmetric unit of the folate complex (but not the 5dfol or ddTHF complexes), supporting the hypothesis that N5 protonation of bound substrate, an important step of the DHFR reaction, occurs by way of such a water molecule. There is no indication of a hydrogen bond between N8 of 5dfol and the backbone carbonyl of Ile-5, suggesting that the bacterial enzyme, unlike the human enzyme [Davies, J. F., II, Delcamp, T. J., Prendergast, N. J., Ashford, V. A., Freisheim, J. H., & Kraut, J. (1990) Biochemistry 29, 9467-9479], does not favor protonation at N8. Perhaps this explains why bacterial DHFR is much less effective than vertebrate DHFR in folate reduction. When the ecDHFR.NADPH complex (space group P3221; M. R. Sawaya, in preparation) is superimposed on the folate and 5dfol complexes, the distances from pteridine C6 to nicotinamide C4 were found to be 2.9 and 2.8 A, respectively, in close agreement with the theoretically calculated optimal distance in the transition state for hydride transfer [Wu, Y. D., & Houk, K. N. (1987) J. Am. Chem. Soc. 109, 906-908, 2226-2227]. In contrast to the planar ring system of folate or 5dfol, the reduced pteridine ring of ddTHF is severely puckered and bent toward the nicotinamide pocket, with the reduced pyridine ring assuming a half-chair type of conformation. This change in shape causes the pteridine ring to bind with O4 closer to Trp-22(N epsilon 1) by over 0.5 A, so that an invariant water molecule now bridges these two atoms with ideal hydrogen bonds. Furthermore, while the pABA rings of folate and 5dfol are nearly coincident and closer to the alpha C helix than to the alpha B helix, those of MTX and ddTHF are displaced along the binding crevice by approximately 1.1 and 0.6 A, respectively, and are equidistant from alpha B and alpha C.(ABSTRACT TRUNCATED AT 400 WORDS)
...
PMID:Isomorphous crystal structures of Escherichia coli dihydrofolate reductase complexed with folate, 5-deazafolate, and 5,10-dideazatetrahydrofolate: mechanistic implications. 787 54


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---