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)

It has been shown in this and other laboratories that during the unfolding of a number of enzymes inactivation generally precedes global unfolding of the enzyme molecule, leading to the suggestion that enzyme active sites are usually more "fragile" and more easily "perturbed" than the molecule as a whole and are therefore conformationally more flexible than the rest of the molecule. However, the role of active site flexibility in enzyme catalysis still remains to be explored. In the induced fit hypothesis originally proposed by Koshland, the presence of the substrate induces a conformational change at the active site so as to fit with the structure of the substrate. By X-ray crystallographic structural analysis of E. coli dihydrofolate reductase liganded with cofactors and substrates, Sawaya and Kraut showed the enzyme in different conformational states indeed while complexed with different ligands, suggesting that the enzyme molecule passes through different conformational states through the whole process of catalysis. Muscle lactate dehydrogenase can be stabilized either in concentrated ammonium sulfate or by cross-linking with glutaraldehyde together with a decrease in enzyme activity which can be restored to the original level in dilute guanidine hydrochloride possibly by increased flexibility at the active site. It is known that a number of enzymes can be activated by chaotropic agents such as urea or guanidine hydrochloride. The activation of dihydrofolate reductase by either urea or guanidine hydrochloride is accompanied by an increase in susceptibility to proteolysis. Isolation of the tryptic peptides of the activated enzyme and sequence analysis allowed identification of the sites of proteolysis to be at or near the active site of the enzyme, indicating an opening up of the active site conformation in the activated state. All the above indicate that active site flexibility plays an important role in enzyme catalysis. It is possible that during the catalytic cycle, the enzyme molecule passes through different stages and each stage requires the molecule to be in a different conformation, especially at the active site. Rapid transition between the different conformational states, and hence the flexibility of the active site, is therefore mandatory for the maximal expression of enzyme activity.
...
PMID:The role of active site flexibility in enzyme catalysis. 952 22

To elucidate the role of a flexible loop (residues 142-149) in the stability and function of Escherichia coli dihydrofolate reductase, alanine-145 in this loop was substituted by site-directed mutagenesis with ten amino acids (Glu, Phe, Gly, His, Ile, Leu, Arg, Ser, Thr, and Val). The amount of three mutant proteins (A145E, A145I, and A145L) in cells was too small to allow the measurement of circular dichroism (CD) spectra and urea unfolding. The CD spectra of other seven mutants were identical with those of the wild-type DHFR, indicating that the native conformation of DHFR was not affected by the mutations. The free energy change of unfolding by urea decreased with an increase in the hydrophobicity of amino acid residues introduced, A145T>A145R>A145G>=A145S>=A145H>A145V++ +>wild-type>=A145F. The steady-state kinetic parameters for the enzyme reaction, Km and ksub, were only slightly influenced by the mutations. These results suggest that site 145 in the flexible loop plays an important role in the stability but has little or no effect on the native structure and function of this enzyme. The characteristics of the mutations are discussed in comparison with those of mutations at site 67 [Ohmae et al. (1996) J. Biochem. 119, 703-710] and at site 121 [Gekko et al. (1994) J. Biochem. 116, 34-41] in two other flexible loops.
...
PMID:Effects of point mutations at the flexible loop alanine-145 of Escherichia coli dihydrofolate reductase on its stability and function. 956 14

We have overexpressed the gene for dihydrofolate reductase (DHFR) from Thermotoga maritima in Escherichia coli and characterized the biochemical properties of the recombinant protein. This enzyme is involved in the de novo synthesis of deoxythymidine 5'-phosphate and is critical for cell growth. High levels of T. maritima DHFR in the new expression system conferred resistance to high levels of DHFR inhibitors which inhibit the growth of non-recombinant cells. The enzyme was purified to homogeneity in the following two steps: heat treatment followed by affinity chromatography or cation-exchange chromatography. Most of the biochemical properties of T. maritima DHFR resemble those of other bacterial or eukaryotic DHFRs, however, some are unique to T. maritima DHFR. The pH optima for activity, Km for substrates, and polypeptide chain length of T. maritima DHFR are similar to those of other DHFRs. In addition, the secondary structure of T. maritima DHFR, as measured by circular dichroism, is similar to that of other DHFRs. Interestingly, T. maritima DHFR exhibits some characteristics of eukaryotic DHFRs, such as a basic pI, an excess of positively charged residues in the polypeptide chain and activation of the enzyme by inorganic salts and urea. Unlike most other DHFRs which are monomeric or part of a bifunctional DHFR-thymidylate synthase (TS) enzyme, T. maritima DHFR seems to generally form a dimer in solution and is also much more thermostable than other DHFRs. It may be that dimer formation is a key factor in determining the stability of T. maritima DHFR.
...
PMID:Purification and characterization of recombinant Thermotoga maritima dihydrofolate reductase. 973 2

hTom20 is an outer mitochondrial membrane receptor involved in protein translocation. The cytosolic domain (aa30-145) and selected truncated versions of this domain were overexpressed and purified to study the structure-function relationship of this protein. Our studies reveal that the secondary structure of the cytosolic domain is very resistant to unfolding by guanidine-HCl and urea and is stabilized mainly by hydrophobic interactions. However, the tertiary structure of the N-terminal targeting signal binding domain (aa30-90) is more flexible. The first 30 amino acids of the cytosolic domain (aa30-60) are involved in recognizing N-terminal targeting signals and in stabilizing the cytosolic domain on the lipid surface. Moreover, we show that specifically aa30-48 interact with the membrane surface; a construct containing aa48-145 will only bind to the membrane surface in the presence of an N-terminal targeting signal peptide. The C-terminal region of hTom20 (aa141-145) interacts with the N-terminal region of hTom20, helping to stabilize the proper conformation of the N-terminal targeting signal binding domain. Finally, hTom20 interacts with the N-terminal targeting signal of preornithine carbamyl transferase fused to dihydrofolate reductase very weakly (Kd = 8 microM), as would be expected if this interaction was the first in a series orchestrated by the import receptor complex to draw the targeted protein into the mitochondrion.
...
PMID:Characterization of the N-terminal targeting signal binding domain of the mitochondrial outer membrane receptor, Tom20. 974 10

An important consideration in the construction of active and stable circularly permuted proteins is the connective sequence that links the native N- and C-termini. For this reason, various lengths of polyglycine linkers (two, three, four, five and six glycines) were employed to connect the original N- and C-termini of a circularly permuted construct of Escherichia coli dihydrofolate reductase (DHFR) in which the new N-terminus was Met16. Examination of the circular dichroism (CD) spectra, gel-filtration chromatography elution profiles, urea-induced unfolding properties and enzyme kinetics revealed that, among the linkers tested, a linker length of five glycines was the most favorable. The Vmax of the circularly permuted variant with a five glycine linker (cpM16G5) was about 20% that of wild-type DHFR, although far UV CD spectra, gel filtration elution time, conformational stability and Km for the substrate dihydrofolate and Kd for the coenzyme NADPH were comparable in the two proteins. Another circularly permuted DHFR with a five glycine linker in which a new N-terminus was created at Leu24 (cpL24G5) was also constructed and assayed. The Vmax of cpL24G5 was almost the same as the wild-type, presumably due to the optimization of the glycine linker. The improved activity of the Leu24 permutant is probably due to the disruption of a catalytically important structure, the M20 loop, in the Met16 permutant.
...
PMID:Effects of the length of a glycine linker connecting the N-and C-termini of a circularly permuted dihydrofolate reductase. 974 24

Proteins appear to contain structural elements which determine the folded structure. If such elements are present, the order of structural elements in the primary structure, i.e. the chain topology, can be disregarded for building of the folded tertiary structure, when they are properly connected to each other by proper linkers. To experimentally examine this, "topological" mutants (designated as GHF33 and GHF34) of Escherichia coli dihydrofolate reductase (DHFR) were designed and constructed by switching two amino acid sequence parts containing the betaF strand and betaG-betaH strands in the primary sequence. In this way, the chain topology of wild-type DHFR, betaA-->alphaB-->betaB-->alphaC-->betaC--> betaD-->alphaE-->betaE-->al phaF-->betaF-->betaG-->betaH, was changed to betaA-->alphaB-->betaB-->alphaC-->betaC--> betaD-->alphaE-->betaE-->al phaF-->betaG-->betaH-->betaF. Such topological mutant proteins were stably expressed and accumulated in E. coli cells, and highly purified. Molecular mass measurements of the purified proteins and their proteolytic fragments confirmed that GHF33 and GHF34 had the designed sequence. In terms of kcat, the GHF33 and GHF34 proteins showed about 10 and 20% of the DHFR activity of the wild-type with Km values of 3.3 microM (GHF33) and 2.1 microM (GHF34), respectively. The topological mutants showed a cooperative two-state transition in urea-induced unfolding experiments with DeltaGH2O values of 4.0 and 4.8 kcal/mol. Whereas, the Km and DeltaGH2O values for wild-type DHFR were 0.9 microM and 6.1 kcal/mol, respectively. The significance of the topological mutations was discussed with respect to protein folding and protein evolution.
...
PMID:Topological mutation of Escherichia coli dihydrofolate reductase. 975 22

To address the effects of single amino acid substitutions on the flexibility of Escherichia coli dihydrofolate reductase (DHFR), the partial specific volume (v(o)) and adiabatic compressibility (beta(s)(o)) were determined for a series of mutants with amino acid replacements at Gly67 (7 mutants), Gly121 (6 mutants), and Ala145 (5 mutants) located in three flexible loops, by means of precise sound velocity and density measurements at 15 degrees C. These mutations induced large changes in v(o) (0.710-0.733 cm(3). g(-1)) and beta(s)(o) (-1.8 x 10(-6)-5.5 x 10(-6) bar(-1)) from the corresponding values for the wild-type enzyme (v(o)=0.723 cm(3). g(-1), beta(s)(o) = 1.7 x 10(-6) bar(-1)), probably due to modifications of internal cavities. The beta(s)(o) value increased with increasing v(o), but showed a decreasing tendency with the volume of the amino acid introduced. There was no significant correlation between beta(s)(o) and the overall stability of the mutants determined from urea denaturation experiments. However, a mutant with a large beta(s)(o) value showed high enzyme activity mainly due to an enhanced catalytic reaction rate (k(cat)) and in part due to increased affinity for the substrate (K(m)), despite the fact that the mutation sites are far from the catalytic site. These results demonstrate that the flexibility of the DHFR molecule is dramatically influenced by a single amino acid substitution in one of these loops and that the flexible loops of this protein play important roles in determining the enzyme function.
...
PMID:Single amino acid substitutions in flexible loops can induce large compressibility changes in dihydrofolate reductase. 1087 54

The thermodynamic and spectroscopic properties of a cysteine-free variant of Escherichia coli dihydrofolate reductase (AS-DHFR) were investigated using the combined effects of urea and temperature as denaturing agents. Circular dichroism (CD), absorption, and fluorescence spectra were recorded during temperature-induced unfolding at different urea concentrations and during urea-induced unfolding at different temperatures. The first three vectors obtained by singular-value decomposition of each set of unfolding spectra were incorporated into a global analysis of a unique thermodynamic model. Although individual unfolding profiles can be described as a two-state process, a simultaneous fit of 99 vectors requires a three-state model as the minimal scheme to describe the unfolding reaction along both perturbation axes. The model, which involves native (N), intermediate (I), and unfolded (U) states, predicts a maximum apparent stability, DeltaG degrees (NU), of 6 kcal mol(-)(1) at 15 degrees C, an apparent m(NU) value of 2 kcal mol(-)(1) M(-)(1), and an apparent heat capacity change, DeltaC(p)()(-NU), of 2.5 kcal mol(-)(1) K(-)(1). The intermediate species has a maximum stability of approximately 2 kcal mol(-)(1) and a compactness closer to that of the native than to that of the unfolded state. The population of the intermediate is maximal ( approximately 70%) around 50 degrees C and falls below the limits of detection of > or =2 M urea or at temperatures of <35 or >65 degrees C. The fluorescence properties of the equilibrium intermediate resemble those of a transient intermediate detected during refolding from the urea-denatured state, suggesting that a tryptophan-containing hydrophobic cluster in the adenosine-binding domain plays a key role in both the equilibrium and kinetic reactions. The CD spectroscopic properties of the native state reveal the presence of two principal isoforms that differ in ligand binding affinities and in the packing of the adenosine-binding domain. The relative populations of these species change slightly with temperature and do not depend on the urea concentration, implying that the two native isoforms are well-structured and compact. Global analysis of data from multiple spectroscopic probes and several methods of unfolding is a powerful tool for revealing structural and thermodynamic properties of partially and fully folded forms of DHFR.
...
PMID:Multistate equilibrium unfolding of Escherichia coli dihydrofolate reductase: thermodynamic and spectroscopic description of the native, intermediate, and unfolded ensembles. 1092 51

Kinetic studies of chicken liver dihydrofolate reductase (CL-DHFR) and Chinese hamster ovary DHFR (CH-DHFR) activated following p-hydroxymercuribenzoate (p-HMB) modification indicate a conformational change at the active site, suggesting a loosening of the enzyme structure upon SH modification. In the present study, limited proteolysis was applied to detect the subtle conformational changes in SH-modified DHFRs. The digested peptide fragments were separated by Tricine SDS-PAGE and sequenced by Edman auto-degradation. The thiol modifier N-iodoacetyl-N'-(5-sulfo-1-nophthyl) ethylenediamine (IAEANS), which activates these DHFRs only weakly, was used as a control. The results of sequencing showed that compared to native enzyme, there is one additional cleavage site near the active site in p-HMB-modified CL-DHFR, two additional sites in p-HMB-modified CH-DHFR, but no additional site for IAEANS-modified DHFRs. These results indicate that activation of DHFRs following thiol modification is accompanied by a conformational change at or near the active site. This subtle change in the active site conformation results in a pronounced change in enzyme activity. This provides further evidence that flexibility at the active site is essential for full expression of enzyme catalytic activity. Comparing results obtained from previous experiments on guanidine- and urea-activated CL-DHFR, this shows that a conformational change near helix(28-39) is sufficient for full activation of DHFR.
...
PMID:Conformational change of dihydrofolate reductase near the active site after thiol modification: detected by limited proteolysis. 1096 90

Dihydrofolate reductase (DHFR) (5,6,7,8-THF: NADDP+ oxidoreductase, EC 1.5.1.3) was purified 205-fold to apparent homogeneity from the crude extracts of Lactobacillus leichmannii. It has UV absorption maxima at 280 nm, M(r) of 20,000, Stokes radius of 0.34 nm and a S20.w value of 0.12 S. The preparation showed the presence of 168 amino acid residues with threonine and lysine as the NH2- and COOH- terminal end-groups respectively and a single reactive sulfhydryl group. pCMB inhibited the enzyme activity (IC50 = 2 microM). The enzyme has a pH optimum of 7.4 and is thermally inactivated at > 35 degrees C. It is activated by 0.1 M KCl and KI and 2 M urea. 3-4 M urea completely inactivated the enzyme. Enzyme has Km values of 3.5 microM and 6.2 microM for NADPH and DHF respectively, and a Ki value of 7 nM for MTX, the inhibition being competitive.
...
PMID:Purification and characterization of dihydrofolate reductase from Lactobacillus leichmannii. 1098 23


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

---