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)

We have made multiple replacements (alanine, arginine, cysteine, histidine, isoleucine, serine, tyrosine) of valine-75 in dihydrofolate reductase from Escherichia coli to examine the relative importance to protein folding of the position that is substituted and the specific character of the amino acid replacement. Valine-75 is part of the eight-stranded beta sheet that forms the structural core of the protein. The isopropyl side chain participates in van der Waals interactions with a number of nonpolar residues, helping to establish a large hydrophobic cluster. Equilibrium studies showed that arginine, histidine, isoleucine, serine, and tyrosine destabilize the protein by 1.9-2.8 kcal mol-1. Alanine and cysteine substitutions have little or no effect. Contrary to other recent studies of the effect of multiple replacements at a hydrophobic site, there is no observed correlation between the changes of the free energy of folding and the changes of the free energy of transfer for the individual amino acids from water to an organic solvent when they are inserted into this site. The effects observed in kinetic studies are both consistent with and extend the equilibrium results; these data indicate that position 75 participates in a rate-limiting step of folding. Some of the equilibrium and kinetic properties of the tyrosine-75 mutant deviated significantly from those of wild-type protein and the other mutants at position 75. (1) The tyrosine variant displayed a complex banding pattern when analyzed by native gel electrophoresis; the wild-type protein and all other mutants at position 75 migrated as single, discrete bands. (2) Comparison of the difference ultraviolet and circular dichroism transition curves showed that a third species is populated at equilibrium; the wild-type protein and all other mutants at position 75 follow a two-state model involving only native and unfolded forms. (3) A third kinetic phase appeared in the unfolding reaction; the wild-type protein and all other mutants at position 75 only showed two kinetic phases in unfolding. Properties 1 and 3 suggest that the tyrosine mutation significantly alters the distribution of native conformers in the protein. These effects on the equilibrium and kinetic data readily display an overriding pattern: residues that would require hydrogen bonding or lead to an expansion of the tightly packed hydrophobic environment in which valine-75 resides destabilize the protein and alter relaxation times of kinetic phases in a consistent manner.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Effects of multiple replacements at a single position on the folding and stability of dihydrofolate reductase from Escherichia coli. 265 2

Recently, the simian type 1 transforming growth factor beta (TGF-beta 1) cDNA was expressed at high levels in Chinese hamster ovary (CHO) cells by dihydrofolate reductase-induced gene amplification (L.E. Gentry, N.R. Webb, G.J. Lim, A.M. Brunner, J.E. Ranchalis, D.R. Twardzik, M.N. Lioubin, H. Marquardt, and A.F. Purchio, Mol. Cell. Biol. 7:3418-3427, 1987). We have now purified and characterized the recombinant proteins released by these cells. Analyses of the precursor proteins by amino acid sequencing identified potentially important proteolytic processing sites. Signal peptide cleavage occurs at the Gly-29-Leu-30 peptide bond of pre-pro-TGF-beta 1, yielding pro-TGF-beta 1 (30 to 390). In addition, proteolytic processing of the precursor to yield mature TGF-beta 1 occurs at the dibasic cleavage site immediately preceding Ala-279, indicating that CHO cells possess the appropriate processing enzyme. Greater than 95% of the biological activity detected in the conditioned medium of the CHO transfectant was due to mature, properly processed growth factor. Highly purified recombinant TGF-beta 1 had the same specific biological activity as natural TGF-beta 1. The concentration of TGF-beta 1 required for half-maximal inhibition of Mv1Lu mink lung epithelial cell growth was approximately 1 to 2 pM. Purified precursor inhibited mink lung cell proliferation at 50 to 60 pM concentrations. The purified precursor preparation was shown to consist of pro-TGF-beta 1 (30 to 390), the pro region of the precursor (30 to 278), and mature TGF-beta 1 (279 to 390) interlinked by at least one disulfide bond with the pro portion of the precursor. These recombinant forms of TGF-beta1 should prove useful for further structural and functional studies.
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PMID:Molecular events in the processing of recombinant type 1 pre-pro-transforming growth factor beta to the mature polypeptide. 318 45

Three mutations of the enzyme dihydrofolate reductase were constructed by oligonucleotide-directed mutagenesis of the cloned Escherichia coli gene. The mutations--at residue 27, aspartic acid replaced with asparagine; at residue 39, proline replaced with cysteine; and at residue 95, glycine replaced with alanine--were designed to answer questions about the relations between molecular structure and function that were raised by the x-ray crystal structures. Properties of the mutant proteins show that Asp-27 is important for catalysis and that perturbation of the local structure at a conserved cis peptide bond following Gly-95 abolishes activity. Substitution of cysteine for proline at residue 39 results in the appearance of new forms of the enzyme that correspond to various oxidation states of the cysteine. One of these forms probably represents a species cross-linked by an intrachain disulfide bridge between the cysteine at position 85 and the new cysteine at position 39.
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PMID:Directed mutagenesis of dihydrofolate reductase. 635 60

Tetrahydrobiopterin, the natural pteridine cofactor for aromatic amino acid hydroxylases, was produced stereopecifically with reference to the C-6 chiral center from 7,8-dihydrobiopterin by the action of dihydrofolate reductase. Similarly, 7,8-dihydro-6-methylpterin was reduced to tetrahydro-6-methylpterin having the same 6-configuration by the same enzyme. The absolute configuration of these tetrahydropterins at the C-6 chiral center was determined to be L, as in (2S)-1,2,3,4-tetrahydro-2-methylquinoxaline, which was servied from L-alanine, by comparison of the CD spectra.
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PMID:Studies on biologically active pteridines. III. The absolute configuration at the C-6 chiral center of tetrahydrobiopterin cofactor and related compounds. 739 Sep 72

Amino acid sequences in proteins can contain residues which complicate biochemical, biophysical, or protein engineering studies but which are not essential for folding or activity. Their replacement with other naturally-occurring amino acids which are not subject to such complications but which maintain essential properties of the protein is a desirable goal. A simple strategy for testing various mutants for their suitability is described for a pair of cysteine residues in dihydrofolate reductase (DHFR) from Escherichia coli. Using a reconstructed gene which preserves the amino acid sequence and introduces a variety of unique restriction sites, the cysteines at positions 85 and 152 were replaced by site-directed and cassette mutagenesis. The enzymatic activity, stability, and folding mechanism of six double mutant DHFR proteins were examined with the purpose of identifying a suitable alternative to wild type DHFR. The Cys85-->Ala and Cys152-->Ser double mutant DHFR was found to retain the four channel folding mechanism and have activity and stability which are comparable to the wild type enzyme. The replacement of the cysteines improved the resistance of DHFR to the irreversible loss of activity at high temperature.
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PMID:A strategy for testing the suitability of cysteine replacements in dihydrofolate reductase from Escherichia coli. 762 11

The gene for the chromosomally encoded dihydrofolate reductase (DHFR) of Staphylococcus epidermidis ATCC 14990 has been cloned and characterized. The structural gene encodes a polypeptide of 161 amino acid residues with a calculated molecular weight of 18,417. This trimethoprim-sensitive (Tmps) DHFR, SeDHFR, differs in only three amino acids (Val-31-->Ile, Gly-43-->Ala, and Phe-98-->Tyr) from the trimethoprim-resistant (Tmpr) S1 DHFR encoded by transposon Tn4003. Since in addition the S. epidermidis gene also forms part of an operon with thyE and open reading frame 140 as in Tn4003, the chromosomally located gene encoding the Tmps SeDHFR is likely to be the molecular origin of the plasmid-located gene encoding the Tmpr S1 DHFR. Site-directed mutagenesis and kinetic analysis of the purified enzymes suggest that a single Phe-->Tyr change at position 98 is the major determinant of trimethoprim resistance.
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PMID:Characterization of the gene for the chromosomal dihydrofolate reductase (DHFR) of Staphylococcus epidermidis ATCC 14990: the origin of the trimethoprim-resistant S1 DHFR from Staphylococcus aureus? 776 89

To elucidate the role of a flexible loop in the stability and function of Escherichia coli dihydrofolate reductase, glycine-121 in a flexible loop (residues 117-131), separated by 19 A from active site Asp27, was substituted by site-directed mutagenesis with eight amino acids (Ala, Val, Leu, Asp, Ser, Cys, Tyr, and His). The free energy change of unfolding decreased in the order of G121A > G121D > G121C > G121S, wild-type > G121H > G121Y > G121L > G121V. The thermal denaturation temperature decreased with all mutations, accompanied by a decrease in the calorimetric enthalpy of denaturation. The steady-state kinetic parameter for the enzyme reaction, Km, was only slightly influenced, but kcat was significantly decreased by the mutations, there being 3- (G121C) to 42-fold (G121L) decreases in kcat/Km compared to that of the wild-type enzyme. The effects of mutations on the stability and enzyme activity were statistically examined as a function of the hydrophobicity and volume of amino acids introduced. The diminished stability and activity with increases in the hydrophobicity and volume of amino acids suggest that the main effect of the mutations would be modification of the flexibility of the loop due to overcrowding of the bulky side chains, overcoming the enhancement of the hydrophobic interaction.
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PMID:Point mutations at glycine-121 of Escherichia coli dihydrofolate reductase: important roles of a flexible loop in the stability and function. 779 83

The amino acid composition of proteins from mesophilic and extremophilic organisms is commonly assumed to reflect the mechanisms of molecular adaptation to extremes of physical conditions. In this context, halophilic behaviour has been attributed to significantly increased numbers of aspartic and glutamic acid residues. However, extending the analysis to a statistically relevant set of related proteins, dihydrofolate reductase from Halobacterium volcanii, as an example, shows that the increase in negative charge is found to be less significant than other exchanges of amino acids (e.g., Ala, Asn, Arg, Lys, Phe, Ser). Thus, the high water binding capacity of negatively charged residues cannot be unambiguously correlated with the anomalous stability of halophilic proteins. A similar caveat holds for generalizations regarding the thermal stability of proteins. In this case, D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was compared with a number of mesophilic and moderately thermophilic homologs. Again, 'traffic rules of stabilization', in terms of amino acid changes in going from mesophilic to thermophilic proteins, cannot be given.
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PMID:Relevance of sequence statistics for the properties of extremophilic proteins. 790 11

A mutant of Lactobacillus casei dihydrofolate reductase has been constructed in which Thr63, a residue which interacts with the 2'-phosphate group of the bound coenzyme, is replaced by alanine. This substitution does not affect kcat, but produces an 800-fold increase in the Km for NADPH, which reflects dissociation of NADPH from the enzyme-NADPH-tetrahydrofolate complex, and a 625-fold increase (corresponding to 3.8 kcal/mol) in the dissociation constant for the enzyme-NADPH complex. The difference in magnitude of these effects indicates a small effect of the substitution on the negative cooperativity between NADPH and tetrahydrofolate. Stopped-flow studies of the kinetics of NADPH binding show that the weaker binding arises predominantly from a decrease in the association rate constant. NMR spectroscopy was used to compare the structures of the mutant and wild-type enzymes in solution, in their complexes with methotrexate and with methotrexate and NADPH. This showed that only minimal structural changes result from the mutation; a total of 47 residues were monitored from their resolved 1H resonances, and of these nine in the binary complex and six in the ternary differed in chemical shift between mutant and wild-type enzyme. These affected residues are confined to the immediate vicinity of residue 63. There is a substantial difference in the 31P chemical shift of the 2'-phosphate of the bound coenzyme, reflecting the loss of the interaction with the side chain of Thr63. The only changes in nuclear Overhauser effects (NOEs) observed were decreases in the intensity of NOEs between protons of the adenine ring of the bound coenzyme and the nearby residues Leu62 and Ile102, showing that the substitution of Thr63 does cause a change in the position or orientation of the adenine ring in its binding site.
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PMID:Effects of substitution of Thr63 by alanine on the structure and function of Lactobacillus casei dihydrofolate reductase. 793 9

Directed mutagenesis has been used to construct five variants of human dihydrofolate reductase in which smaller residues are substituted for phenylalanine 34, a residue participating in the binding of substrate and methotrexate by interaction with their pteridine rings. The variant enzymes are stable and have decreased affinities for methotrexate (by factors of 2700-60000 at pH 7.65) due to a decreased rate of methotrexate association and a much larger increase in the rate constant for dissociation. However, the catalytic efficiencies of the variants are also lowered by factors of 160-5000, so that it is doubtful whether these enzymes are capable of conferring methotrexate resistance on the cells harboring them. High concentrations of dihydrofolate cause marked inhibition of all the variants, which complicates the determination of kinetic parameters. By the use of stopped-flow spectrophotometry and fluorimetry and other methods, it has been shown that, like the wild-type enzyme, the variants have a branched reaction pathway, but in contrast to the wild-type enzyme, the distribution of flux between alternate pathways is dependent on the concentration of dihydrofolate. This different branch point is a consequence of the very rapid dissociation of tetrahydrofolate from the ternary product complexes of the variant enzymes. Inhibition by dihydrofolate is due to its combination with the enzyme-NADP complex and the slow dissociation of NADP from the resulting abortive complex. When steady state kinetics for this model are simulated using the experimentally determined rate and dissociation constants for the alanine 34 variant, most steady state experimental results are closely approximated.
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PMID:Critical role of phenylalanine 34 of human dihydrofolate reductase in substrate and inhibitor binding and in catalysis. 806 Oct 3


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