Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An enzyme which cleaves the benzene ring of 3,5-dichlorocatechol has been purified to homogeneity from Pseudomonas cepacia CSV90, grown with 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole carbon source. The enzyme was a nonheme ferric dioxygenase and catalyzed the intradiol cleavage of all the examined catechol derivatives, 3,5-dichlorocatechol having the highest specificity constant of 7.3 microM-1s-1 in an air-saturated buffer. No extradiol-cleaving activity was observed. Thus, the enzyme was designated as 3,5-dichlorocatechol 1,2-dioxygenase. The molecular weight of the native enzyme was ascertained to be 56,000 by light scattering method, while the M(r) value of the enzyme denatured with 6 M guanidine-HCl or sodium dodecyl sulfate was 29,000 or 31,600, respectively, suggesting that the enzyme was a homodimer. The iron content was estimated to be 0.89 mol per mole of enzyme. The enzyme was deep red and exhibited a broad absorption spectrum with a maximum at around 425 nm, which was bleached by sodium dithionite, and shifted to 515 nm upon anaerobic 3,5-dichlorocatechol binding. The catalytic constant and the Km values for 3,5-dichlorocatechol and oxygen were 34.7 s-1 and 4.4 and 652 microM, respectively, at pH 8 and 25 degrees C. Some heavy metal ions, chelating agents and sulfhydryl reagents inhibited the activity. The NH2-terminal sequence was determined up to 44 amino acid residues and compared with those of the other catechol dioxygenases previously reported.
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PMID:Purification of 3,5-dichlorocatechol 1,2-dioxygenase, a nonheme iron dioxygenase and a key enzyme in the biodegradation of a herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), from Pseudomonas cepacia CSV90. 767 68

Effect of two classical and potent denaturants, guanidine hydrochloride (GuHCl) and guanidine thiocyanate (GuHSCN) on purified wheat germ lipase has been studied. Lipase was found to be active only up to 5 M GuHCl and 1.5 M GuHSCN. The extent of interaction was determined by the measurement of apparent partial specific volume of the enzyme in presence of these two denaturants. While the preferential interaction parameter (zeta 3) has values of 0.08 +/- 0.02 and 0.14 +/- 0.03 g/g, the interaction parameter (delta m3/delta m2)T,mu 1, mu3 has values of 35 +/- 9 and 50 +/- 10 mole/mole for GuHCl and GuHSCN, respectively. The number of denaturant molecules bound to the enzyme, A3, obtained experimentally were 0.486 +/- 0.020 and 0.348 +/- 0.020 g/g and the calculated values were 0.459 +/- 0.023 and 0.567 +/- 0.030 g/g for 6 M GuHCl and 3 M GuHSCN, respectively. The volume change occurring upon denaturation results in -420 +/- 42 and -462 +/- 84 ml/mole in 6 M GuHCl and 3 M GuHSCN, respectively. The denaturation is accompanied by exposure of hydrophobic groups to the bulk solvent as confirmed by fluorescence emission measurements of the enzyme. The Tm measurements indicated a control value of 56 +/- 1 degree C. In presence of 6 M GuHCl/3 M GuHSCN, the value was 42 +/- 1 degree C. These results explain the retention of lipase activity even at 5 M GuHCl from a mechanistic point of view.
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PMID:Interaction of guanidine hydrochloride and guanidine thiocyanate with wheat germ lipase. 800 14

Under nondenaturing conditions, 1 mol of horse plasma gelsolin reacts with 1.9 +/- 0.5 mol (mean +/- SD, n = 6) of the sulfhydryl-specific fluorescent reagent 6-acryloyl-2-dimethylaminonaphthalene (acrylodan). The degree of labeling in 6 M guanidine-HCl increases to about 3 mol of acrylodan per mole of gelsolin. Viscosity studies show that the modified gelsolin retains its ability to sever F-actin filaments. Circular dichroism spectra in the peptide bond absorption region are indistinguishable for labeled and unmodified gelsolin at room temperature. The thermal stability of gelsolin, as monitored by circular dichroism, is unaffected by reaction with acrylodan. While circular dichroism spectra of acrylodan-labeled gelsolin recorded at room temperature are not influenced significantly by Ca2+, fluorescence studies reveal a number of Ca(2+)-dependent changes in the protein. Ca2+ causes a decrease and red-shift in fluorescence emission, an increase in sensitivity to quenching by I-, and a decrease in polarization of the fluorescence of acrylodan-labeled gelsolin. Together, these changes in fluorescence properties indicate there to be an increased exposure of the label to the solvent when gelsolin binds Ca2+. Fluorescence polarization experiments in which acrylodan-labeled gelsolin is titrated with actin emphasize that Ca2+ is required for these two proteins to interact.
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PMID:Fluorescent responses of acrylodan-labeled plasma gelsolin. 838 3

The folding and stability of maltose binding protein (MBP) have been investigated as a function of pH and temperature by intrinsic tryptophan fluorescence, far- and near-UV circular dichroism, and high-sensitivity differential scanning calorimetric measurements. MBP is a monomeric, two-domain protein containing 370 amino acids. The protein is stable in the pH range of 4-10.5 at 25 degrees C. The protein exhibits reversible, two-state, thermal and guanidine hydrochloride-mediated denaturation at neutral pH. The thermostability of MBP is maximal at pH 6, with a Tm of 64.9 degrees C and a deltaHm of 259.7 kcal mol(-1). The linear dependence of deltaHm on Tm was used to estimate a value of deltaCp of 7.9 kcal mol(-1) K(-1) or 21.3 cal (mol of residue)(-1) K(-1). These values are higher than the corresponding deltaCp's for most globular proteins studied to date. However, the extrapolated values of deltaH and deltaS (per mole of residue) at 110 degrees C are similar to those of other globular proteins. These data have been used to show that the temperature at which a protein undergoes cold denaturation depends primarily on the deltaCp (per mol of residue) and that this temperature increases with an increase in deltaCp. The predicted decrease in stability of MBP at low temperatures was experimentally confirmed by carrying out denaturant-mediated unfolding studies at neutral pH at 2 and 28 degrees C.
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PMID:Thermodynamic characterization of the reversible, two-state unfolding of maltose binding protein, a large two-domain protein. 912 24

Slow refolding of human apolipoprotein E (apoE) in solution after guanidine- or cholate-induced denaturation followed by dialysis under controlled conditions was investigated using various spectroscopic properties of fluorescein- and dansyl-labeled apolipoprotein molecules. The results suggest that the last phase(s) of apoE refolding in solution include a slow (several hours at 24 degrees C) interconversion of a self-associated 'open' conformer into a more dense 'closed' conformer. The hydrophobic interactions are primarily responsible for the formation of this more compact apoE structure. To visualize the contribution of apolipoprotein conformation and/or the number of 'active' lipid-bound apoE molecules in the reaction of binding to the low density lipoprotein receptor (LDLr) by solid-phase binding assay, the complexes of human plasma apolipoprotein or recombinant (rec) apoE3 with dipalmitoylphosphatidylcholine (DPPC) or palmitoyloleoylphosphatidylcholine (POPC) varying in size were used. For seven complexes with plasma protein (four DPPC and three POPC complexes), the final phosphatidylcholine (PC)/protein mole ratio ranged from 117 to 279; affinity constant K(a) averaged for both PCs and plotted against this ratio abruptly increased from 3.8 x 10(7) to 3.8 x 10(8) M(-1) with a transition midpoint of 150-180 PC/apoE, mole ratio. Two DPPC complexes with rec protein bind much more efficiently. Complexes with both plasma and rec apoE were able to compete with very low density lipoproteins (VLDL) or low density lipoproteins (LDL) isolated from patients with E3/3 phenotype, for binding to the LDLr. Again, the competition efficiency abruptly increased at the increase in PC content with a transition midpoint of 130 PC/apoE, mole ratio. The transitions observed both in direct and competitive binding assay probably correspond to the abrupt increase in the number of 'active' apoE molecules on the complex surface accompanying the change in the size and/or in the shape of the complexes. The efficiency of apoE and apoB as the corresponding major ligands in the binding reaction of VLDL and LDL to the LDL receptor was compared. VLDL bind to LDLr following a simple encounter complex model, while LDL binding was characterized by a more complex two-step model with an additional isomerization step. The analysis of the binding data led us to suggest the existence of the continuum from several (2-3) apoE molecules on the surface of TG-rich particles that resulted in the increased binding affinity, on average 3.5-fold higher, compared to LDL. The existence of a complex equilibrium between aqueous and different lipid-bound forms of apoE is proposed, in particular, the formation of a transient disc-lipoprotein particle structure during the interaction with LDLr in vivo as well as in LPL-stimulated lipolysis of the lipid phase of the particle.
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PMID:Conformation of apolipoprotein E both in free and in lipid-bound form may determine the avidity of triglyceride-rich lipoproteins to the LDL receptor: structural and kinetic study. 1068 27

The ferredoxin from the thermophilic archaeon Acidianus ambivalens is a small monomeric seven-iron protein with a thermal midpoint (T(m)) of 122 degrees C (pH 7). To gain insight into the basis of its thermostability, we have characterized unfolding reactions induced chemically and thermally at various pHs. Thermal unfolding of this ferredoxin, in the presence of various guanidine hydrochloride (GuHCl) concentrations, yields a linear correlation between unfolding enthalpies (DeltaH[T(m)]) and T(m) from which an upper limit for the heat capacity of unfolding (DeltaC(P)) was determined to be 3.15 +/- 0.1 kJ/(mole * K). Only by the use of the stronger denaturant guanidine thiocyanate (GuSCN) is unfolding of A. ambivalens ferredoxin at pH 7 (20 degrees C) observed ([GuSCN](1/2) = 3.1 M; DeltaG(U)[H(2)O] = 79 +/- 8 kJ/mole). The protein is, however, less stable at low pH: At pH 2.5, T(m) is 64 +/- 1 degrees C, and GuHCl-induced unfolding shows a midpoint at 2.3 M (DeltaG(U)[H(2)O] = 20 +/- 1 kJ/mole). These results support that electrostatic interactions contribute significantly to the stability. Analysis of the three-dimensional molecular model of the protein shows that there are several possible ion pairs on the surface. In addition, ferredoxin incorporates two iron-sulfur clusters and a zinc ion that all coordinate deprotonated side chains. The zinc remains bound in the unfolded state whereas the iron-sulfur clusters transiently form linear three-iron species (in pH range 2.5 to 10), which are associated with the unfolded polypeptide, before their complete degradation.
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PMID:High stability of a ferredoxin from the hyperthermophilic archaeon A. ambivalens: involvement of electrostatic interactions and cofactors. 1146 51

We have determined the stability curve of bovine adenosine deaminase via titrations with guanidine hydrochloride at pH = 6.3 from 5 to 65 degrees C. The data indicate that the enzyme undergoes an abrupt conformational transition at approximate, equals 29 degrees C, a finding supported by a temperature scan of the intrinsic enzyme fluorescence emission. Analysis of the data above and below this temperature with the modified Gibbs-Helmholtz equation allows for complete description of the equilibrium unfolding thermodynamics for either enzyme conformation. The high-temperature form of the enzyme is described by DeltaH degrees = 648 +/- 37 kJ/mole, DeltaC(P) = 23.2 +/- 2.5 kJ/mole-K, and a heat denaturational temperature T(h)(dn) = 72.5 +/- 0.9 degrees C. The low-temperature form is described by DeltaH degrees = 1284 +/- 47 kJ/mole, DeltaCP = 73.2 +/- 4.9 kJ/mole-K, and T(h)(dn) = 32.6 +/- 0.6 degrees C. Further thermodynamic analysis of the conformations that predominate at 38.3 degrees C, the bovine normal body temperature, and at 4 degrees C, where the crystals for x-ray structural analyses were formed, suggest that the stability of either form is due to favorable amino acid side chain nonpolar interactions with these interactions being much more optimized in the low-temperature conformation. We therefore conclude that the structure as determined by x-ray crystallographic methods cannot be the physiological structure. The data also suggest that the general calculation of enzyme stability curves from the extrapolation of heat denaturation data may inaccurately represent the enzyme stability as a low-temperature, nondenaturational transition is assumed not to exist. Further consequences in terms of general enzyme catalysis are also discussed.
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PMID:The stability curve of bovine adenosine deaminase is bimodal. 1243 75

Spectrophotometric profiles representing the unfolding induced by guanidine on Bothrops moojeni myotoxins-I (MjTX-I) and II (MjTX-II), Bothrops jararacussu bothropstoxin-I (BthTX-I) and Bothrops pirajai piratoxin-I (PrTX-I) were obtained and compared with those obtained with bovine ribonuclease A (RNAse) and trypsin. The molar (epsilon(1M)) and percent (epsilon(1%)) extinction coefficients were determined for the four myotoxins as well as for RNAse and trypsin as reference parameters. These coefficients were then used throughout this work. The changes in free energy (deltaGD(H)(2)(O)) corresponding to zero guanidine concentration and the guanidine concentrations (D(1/2)) able to convert 50% of the molecules from the native to the unfolded state were determined. The values of deltaGD (H)(2)(O) ranged from 4.42 (BthTX-I) to 8.02 (MjTX-I) kcal/mole, compared with 6.47 and 6.88 kcal/mole for trypsin and RNAse, respectively. The values for deltaGD(H)(2)(O) and D1/2 showed that BthTX-I is the least stable among the four myotoxins assayed, with a D1/2 close to that of RNAse, while MjTX-II is conformationally the most stable. Monitoring of the unfolding of RNAse and PrTX-I by a 0 to 6 M urea gradient PAGE revealed transitions from the native (N) to the unfolded (U) state with deltaG(N-U)of 0.22 and 0.41 kcal/mole, respectively. Sigmoidal curves showed well-defined two-stage transitions for both proteins.
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PMID:Spectroscopic analysis of the stability of bothrops myotoxic phospholipases A2 to guanidine and urea denaturation. 1262 31

The thermodynamic stabilities of three monomeric variants of the bacteriophage lambda Cro repressor that differ only in the sequence of two amino acids at the apex of an engineered beta-hairpin have been determined. The sequences of the turns are EVK-XX-EVK, where the two central residues are DG, GG, and GT, respectively. Standard-state unfolding free energies, determined from circular dichroism measurements as a function of urea concentration, range from 2.4 to 2.7 kcal/mole, while those determined from guanidine hydrochloride range from 2.8 to 3.3 kcal/mole for the three proteins. Thermal denaturation yields van't Hoff unfolding enthalpies of 36 to 40 kcal /mole at midpoint temperatures in the range of 53 to 58 degrees C. Extrapolation of the thermal denaturation free energies with heat capacities of 400 to 600 cal/mole deg gives good agreement with the parameters determined in denaturant titrations. As predicted from statistical surveys of amino acid replacements in beta-hairpins, energetic barriers to transformation from a type I' turn (DG) to a type II' turn (GT) can be quite small.
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PMID:Stability of monomeric Cro variants: Isoenergetic transformation of a type I' to a type II' beta-hairpin by single amino acid replacements. 1271 34

To understand the structural basis of thermostability, we have determined the solution structure of a thermophilic ribosomal protein L30e from Thermococcus celer by NMR spectroscopy. The conformational stability of T. celer L30e was measured by guanidine and thermal-induced denaturation, and compared with that obtained for yeast L30e, a mesophilic homolog. The melting temperature of T. celer L30e was 94 degrees C, whereas the yeast protein denatured irreversibly at temperatures >45 degrees C. The two homologous proteins also differ greatly in their stability at 25 degrees C: the free energy of unfolding was 45 kJ/mole for T. celer L30e and 14 kJ/mole for the yeast homolog. The solution structure of T. celer L30e was compared with that of the yeast homolog. Although the two homologous proteins do not differ significantly in their number of hydrogen bonds and the amount of solvent accessible surface area buried with folding, the thermophilic T. celer L30e was found to have more long-range ion pairs, more proline residues in loops, and better helix capping residues in helix-1 and helix-4. A K9A variant of T. celer L30e was created by site-directed mutagenesis to examine the role of electrostatic interactions on protein stability. Although the melting temperatures of the K9A variant is approximately 8 degrees C lower than that of the wild-type L30e, their difference in T(m) is narrowed to approximately 4.2 degrees C at 0.5 M NaCl. This salt-dependency of melting temperatures strongly suggests that electrostatic interactions contribute to the thermostability of T. celer L30e.
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PMID:Solution structure and thermal stability of ribosomal protein L30e from hyperthermophilic archaeon Thermococcus celer. 1282 94


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