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

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

The amino acid sequence of a trimethoprim-resistant dihydrofolate reductase (EC 1.5.1.3) specified by the R-plasmid R67 is described. The sequence was deduced from automatic and manual sequence analysis of the intact protein, the fragments produced by cyanogen bromide cleavage, and peptides derived from the largest cyanogen bromide fragment by digestion with trypsin, Staphylococcus aureus V8 proteus, chymotrypsin, and Lysobacter enzymogenes alpha-lytic protease. The complete sequence comprises 78 residues in a single polypeptide chain of molecular weight 8444. No evidence of heterogeneity was obtained, indicating that all subunits of the native enzyme are identical. Comparison of the sequence with that of all known dihydrofolate reductases shows no significant sequence homology.
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PMID:The amino acid sequence of the trimethoprim-resistant dihydrofolate reductase specified in Escherichia coli by R-plasmid R67. 38 58

A new method is presented for calculating a type of quantitative structure-activity relationship, given experimental data on the binding affinity of a series of ligands to a receptor site on a protein. All ligands are presumed to have known chemical structure but may be conformationally flexible, and all are presumed to bind to the same, single, fairly rigid site of the (pure) receptor protein molecule. Given the experimentally determined free energies of binding of the ligand molecules, possible binding sites are deduced in terms of geometry and the chemical character of the various parts of the site. A test of the method is given for a series of chymotrypsin inhibitors and for a series of dihydrofolate reductase inhibitors. The proposed dihydrofolate reductase site suggests that a quinazoline inhibitor may rock between two different binding nodes depending on the pK of the ring N(1).
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PMID:Distance geometry approach to rationalizing binding data. 49 May 43

A comprehensive study on enzyme ligand interactions by QSAR techniques is discussed. Thirteen correlation equations are presented which relate activity of 1086 ligands of isolated chloroplasts, chymotrypsin, dihydrofolate reductase, xanthine oxidase and guanine deaminase to their chemical structures. Two kinds of space within and on the surface of an enzyme are defined by means of pi and MR constants. Emphasis is put on the use of indicator variables as a means of rationalizing special enzyme-ligand interactions. The use of such studies for drug development is discussed.
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PMID:[Correlative analysis in the study of enzyme-ligand interactions]. 73 55

R67 dihydrofolate reductase (DHFR) is a novel protein that provides clinical resistance to the antibacterial drug trimethoprim. The crystal structure of a dimeric form of R67 DHFR indicates the first 16 amino acids are disordered [Matthews et al. (1986) Biochemistry 25, 4194-4204]. To investigate whether these amino acids are necessary for protein function, the first 16 N-terminal residues have been cleaved off by chymotrypsin. The truncated protein is fully active with kcat = 1.3 s-1, Km(NADPH) = 3.0 microM, and Km(dihydrofolate) = 5.8 microM. This result suggests the functional core of the protein resides in the beta-barrel structure defined by residues 27-78. To study this protein further, synthetic genes coding for full-length and truncated R67 DHFRs were constructed. Surprisingly, the gene coding for truncated R67 DHFR does not produce protein in vivo or confer trimethoprim resistance upon Escherichia coli. Therefore, the relative stabilities of native and truncated R67 DHFR were investigated by equilibrium unfolding studies. Unfolding of dimeric native R67 DHFR is protein concentration dependent and can be described by a two-state model involving native dimer and unfolded monomer. Using absorbance, fluorescence, and circular dichroism techniques, an average delta GH2O of 13.9 kcal mol-1 is found for native R67 DHFR. In contrast, an average delta GH2O of 11.3 kcal mol-1 is observed for truncated R67 DHFR. These results indicate native R67 DHFR is 2.6 kcal mol-1 more stable than truncated protein. This stability difference may be part of the reason why protein from the truncated gene is not found in vivo in E. coli.
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PMID:Construction of a synthetic gene for an R-plasmid-encoded dihydrofolate reductase and studies on the role of the N-terminus in the protein. 193 13

We have applied exciton theory to estimate the circular dichroism (CD) contribution of the Trp Bb transition to the far-UV circular dichroism of globular proteins. Strong exciton couplets are predicted for a number of proteins, including dihydrofolate reductase (DHFR), chymotrypsin and chymotrypsinogen. These predicted CD spectra are dominated by the contributions of the closest pair, W47-W74 in DHFR and W174-W215 in chymotrypsinogen. The sign and magnitude of the predicted couplets are consistent with experimental data for DHFR and its W74L mutant, and with the previously unexplained CD changes upon chymotrypsin activation. More extensive coupled-oscillator interactions among all aromatic and peptide chromophores are described for DHFR and barnase. The total far-UV CD spectra predicted for these proteins agree poorly with experiment, primarily owing to difficulties in the calculation of the peptide CD. Nevertheless, difference spectra calculated between the wild-type spectra and those of mutants in which individual aromatic residues are replaced with non-chromophoric side chains show satisfactory agreement with experiment in most cases.
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PMID:Contributions of tryptophan side chains to the circular dichroism of globular proteins: exciton couplets and coupled oscillators. 754 40

Cyclophilin and FK506 binding protein (FKBP) accelerate cis-trans peptidyl-prolyl isomerization and bind to and mediate the effects of the immunosuppressants cyclosporin A and FK506. The normal cellular functions of these proteins, however, are unknown. We altered the active sites of FKBP12 and mitochondrial cyclophilin from the yeast Saccharomyces cerevisiae by introducing mutations previously reported to inactivate these enzymes. Surprisingly, most of these mutant enzymes were biologically active in vivo. In accord with previous reports, all of the mutant enzymes had little or no detectable prolyl isomerase activity in the standard peptide substrate-chymotrypsin coupled in vitro assay. However, in a variation of this assay in which the protease is omitted, the mutant enzymes exhibited substantial levels of prolyl isomerase activity (5-20% of wild-type), revealing that these mutations confer sensitivity to protease digestion and that the classic in vitro assay for prolyl isomerase activity may be misleading. In addition, the mutant enzymes exhibited near wild-type activity with two protein substrates, dihydrofolate reductase and ribonuclease T1, whose folding is accelerated by prolyl isomerases. Thus, a number of cyclophilin and FKBP12 "active-site" mutants previously identified are largely active but protease sensitive, in accord with our findings that these mutants display wild-type functions in vivo. One mitochondrial cyclophilin mutant (R73A), and also the wild-type human FKBP12 enzyme, catalyze protein folding in vitro but lack biological activity in vivo in yeast. Our findings provide evidence that both prolyl isomerase activity and other structural features are linked to FKBP and cyclophilin in vivo functions and suggest caution in the use of these active-site mutations to study FKBP and cyclophilin functions.
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PMID:Functions of FKBP12 and mitochondrial cyclophilin active site residues in vitro and in vivo in Saccharomyces cerevisiae. 936 68

PA700, the 19 S regulatory complex of the 26 S proteasome, plays a central role in the recognition and efficient degradation of misfolded proteins. PA700 promotes degradation by recruiting proteasomal substrates utilizing polyubiquitin chains and chaperone-like binding activities and by opening the access to the core of the 20 S proteasome to promote degradation. Here we provide evidence that PA700 in addition to binding misfolded protein substrates also acts to remodel their conformation prior to proteolysis. Scrambled RNase A (scRNase A), a misfolded protein, only slowly refolds spontaneously into an active form because of the rate-limiting unfolding of misfolded disulfide isomers. Notably, PA700 accelerates the rate of reactivation of scRNase A, consistent with its ability to increase the exposure of these disulfide bonds to the solvent. In this regard, PA700 also exposes otherwise buried sites to digestion by exogenous chymotrypsin in a polyubiquitinated enzymatically active substrate, pentaubiquitinated dihydrofolate reductase, Ub(5)DHFR. The dihydrofolate reductase ligand methotrexate counters the ability of PA700 to promote digestion by chymotrypsin. Together, these results indicate that in addition to increasing substrate affinity and opening the access channel to the catalytic sites, PA700 activates proteasomal degradation by remodeling the conformation of protein substrates.
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PMID:Conformational remodeling of proteasomal substrates by PA700, the 19 S regulatory complex of the 26 S proteasome. 1201 Oct 44

Understanding the molecular mechanisms of enzyme catalysis and allosteric regulation has been a primary goal of biochemistry for many years. The dynamics of these processes, approached through a variety of kinetic methods, are discussed. The results obtained for many different enzymes suggest that multiple intermediates and conformations are general characteristics of the catalytic process and allosteric regulation. Ribonuclease, dihydrofolate reductase, chymotrypsin, aspartate aminotransferase, and aspartate transcarbamoylase are considered as specific examples. Typical and maximum rates of conformational changes and catalysis are also discussed, based on results obtained from model systems. The nature and rates of interconversion of the intermediates, along with structural information, can be used as the bases for understanding the incredible catalytic efficiency of enzymes. Potential roles of conformational changes in the catalytic process are discussed in terms of static and environmental effects, and in terms of dynamic coupling within the enzyme-substrate complex.
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PMID:Multiple conformational changes in enzyme catalysis. 1208 70

The interaction of type II R67 dihydrofolate reductase (DHFR) with its cofactor nicotinamide adenine dinucleotide phosphate (NADP(+)) has been studied using nuclear magnetic resonance (NMR). Doubly labeled [U-(13)C,(15)N]DHFR was obtained from Escherichia coli grown on a medium containing [U-(13)C]-D-glucose and (15)NH(4)Cl, and the 16 disordered N-terminal amino acids were removed by treatment with chymotrypsin. Backbone and side chain NMR assignments were made using triple-resonance experiments. The degeneracy of the amide (1)H and (15)N shifts of the tetrameric DHFR was preserved upon addition of NADP(+), consistent with kinetic averaging among equivalent binding sites. Analysis of the more titration-sensitive DHFR amide resonances as a function of added NADP(+) gave a K(D) of 131 +/- 50 microM, consistent with previous determinations using other methodology. We have found that the (1)H spectrum of NADP(+) in the presence of the R67 DHFR changes as a function of time. Comparison with standard samples and mass spectrometric analysis indicates a slow conversion of NADP(+) to NAD(+), i.e., an apparent NADP(+) phosphatase activity. Studies of this activity in the presence of folate and a folate analogue support the conclusion that this activity results from an interaction with the DHFR rather than a contaminating phosphatase. (1)H NMR studies of a mixture of NADP(+) and NADPH in the presence of the enzyme reveal that a ternary complex forms in which the N-4A and N-4B nuclei of the NADPH are in the proximity of the N-4 and N-5 nuclei of NADP(+). Studies using the NADP(+) analogue acetylpyridine adenosine dinucleotide phosphate (APADP(+)) demonstrated a low level of enzyme-catalyzed hydride transfer from NADPH. Analysis of DHFR backbone dynamics revealed little change upon binding of NADP(+). These additional catalytic activities and dynamic behavior are in marked contrast to those of type I DHFR.
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PMID:NMR studies of the interaction of a type II dihydrofolate reductase with pyridine nucleotides reveal unexpected phosphatase and reductase activity. 1450 65

The PROPKA method for the prediction of the pK(a) values of ionizable residues in proteins is extended to include the effect of non-proteinaceous ligands on protein pK(a) values as well as predict the change in pK(a) values of ionizable groups on the ligand itself. This new version of PROPKA (PROPKA 2.0) is, as much as possible, developed by adapting the empirical rules underlying PROPKA 1.0 to ligand functional groups. Thus, the speed of PROPKA is retained, so that the pK(a) values of all ionizable groups are computed in a matter of seconds for most proteins. This adaptation is validated by comparing PROPKA 2.0 predictions to experimental data for 26 protein-ligand complexes including trypsin, thrombin, three pepsins, HIV-1 protease, chymotrypsin, xylanase, hydroxynitrile lyase, and dihydrofolate reductase. For trypsin and thrombin, large protonation state changes (|n| > 0.5) have been observed experimentally for 4 out of 14 ligand complexes. PROPKA 2.0 and Klebe's PEOE approach (Czodrowski P et al. J Mol Biol 2007;367:1347-1356) both identify three of the four large protonation state changes. The protonation state changes due to plasmepsin II, cathepsin D and endothiapepsin binding to pepstatin are predicted to within 0.4 proton units at pH 6.5 and 7.0, respectively. The PROPKA 2.0 results indicate that structural changes due to ligand binding contribute significantly to the proton uptake/release, as do residues far away from the binding site, primarily due to the change in the local environment of a particular residue and hence the change in the local hydrogen bonding network. Overall the results suggest that PROPKA 2.0 provides a good description of the protein-ligand interactions that have an important effect on the pK(a) values of titratable groups, thereby permitting fast and accurate determination of the protonation states of key residues and ligand functional groups within the binding or active site of a protein.
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PMID:Very fast prediction and rationalization of pKa values for protein-ligand complexes. 1849 3


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