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At least 415 records · Page 23Linked to original sources
PubMed1991

Effects of inhibitors of N-linked oligosaccharide processing on the secretion, stability, and activity of lecithin:cholesterol acyltransferase.

The structure and function of the carbohydrate moiety of human lecithin:cholesterol acyltransferase (LCAT) were determined by using several glycosidases in reaction with the isolated plasma protein or by using specific inhibitors of glycoprotein assembly with cultured cells secreting LCAT activity. Analysis of the plasma enzyme indicated that almost all of the large carbohydrate moiety of LCAT (approximately 25% w/w) was N-linked with part of the high-mannose and part of the complex type. This analysis was confirmed with metabolic inhibitors of carbohydrate processing by using CHO cells stably transfected with the human LCAT gene. Inhibitors of the subsequent processing of the N-linked high-mannose chains formed by glucosidase activity were without effect on either the secretion rate or the catalytic activity of LCAT. The inhibition of catalytic activity by glucosidase inhibitors applied to both the phospholipase and the acyltransferase activities of LCAT. The reduction of the LCAT catalytic rate by terminal glycosidase inhibitors was without effect on apparent Km and did not affect enzyme stability. These data indicate an unusual specific role for high-mannose carbohydrates in the catalytic mechanism of LCAT.

Alkaloids↗
PubMed2005

Modulation of Streptomyces leucine aminopeptidase by calcium: identification and functional analysis of key residues in activation and stabilization by calcium.

Streptomyces griseus leucine aminopeptidase (SGAP), which has two zinc atoms in its active site, is clinically important as a model for understanding the structure and mechanism of action of other metallopeptidases. SGAP is a calcium-activated and calcium-stabilized enzyme, and its activation by calcium correlates with substrate specificity. In our previous study, we found a non-calcium-modulated leucine aminopeptidase secreted by Streptomyces septatus, the primary structure of which showed 71% identity with SGAP. In this study, we constructed chimeras of SGAP and S. septatus aminopeptidase by using an in vivo DNA shuffling system and several mutant enzymes by site-directed mutagenesis to identify the key residues in this modulation by calcium. We identified the key residues Asp-173 and Asp-174 of SGAP associated with both SGAP activation and stabilization by calcium. We also showed that the known calcium-binding site, which is composed of Asp-3, Ile-4, Asp-262, and Asp-266 of SGAP, only contributes to SGAP stabilization by calcium. Furthermore, we identified an important residue, Glu-196, that functions in cooperation with Asp-173, Asp-174, and calcium to increase the catalytic activity of SGAP.

Amino Acid Sequence↗
PubMed2005

PEGylation of yeast cytosine deaminase for pretargeting.

Yeast cytosine deaminase (yCD) was cloned, expressed, and purified by affinity chromatography. We have characterized the products resulting from covalent attachment of 2-4 PEG chains on yCD and determined the major and minor isomers for each respective conjugate. The results show that for non-covalently associated homodimers, it is possible to characterize and deduce PEGylation levels on individual subunits through the concurrent use of size exclusion chromatography (SEC), MALDI-TOF MS, and SDS-PAGE gels. The results also show that contrary to what we expected, attaching more than two PEG chains to yCD decreased its stability. Enzymatic activity studies revealed that the fusion of an N-terminus purification tag on yCD has no significant effect on 5-fluorocytosine or cytosine affinity, with apparent turnover rates remaining within 10(5) M(-1) . s(-1). Stability studies at 37 degrees C revealed that t1/2 = 8-9 h for yCD and 2mPEG(5K)-yCD, whereas for 3-, 4mPEG(5K)-yCD and yCD/BSA, t(1/2) < 2 h. Incubation of BSA with yCD also decreased enzyme stability over prolonged incubation at 37 degrees C. This finding is important if yCD is to be used in a pretargeting strategy.

Cloning, Molecular↗
PubMed1999

Guinea pig liver transglutaminase: A modified purification procedure affording enzyme with superior activity in greater yield.

Tissue transglutaminase purified from guinea pig livers has a very broad substrate specificity in comparison with other members of the transglutaminase family and therefore is useful for substrate analogue kinetic studies. Modifications made in our laboratory to the standard purification protocol (J. E. Folk and S. I. Chung, 1985, Methods Enzymol. 113, 358-364) have yielded a 28% increase in specific activity and 55% increase in overall yield, while reducing the number of steps to the purification. Herein we report some of the highest yields and specific activities for guinea pig liver transglutaminase found in the literature, as well as the use of lyophilization as a solution to the long-standing problem of enzyme stability during storage.

Animals↗
PubMed2006

Expression of a laccase cDNA from Trametes sp. AH28-2 in Pichia pastoris and mutagenesis of transformants by nitrogen ion implantation.

A laccase cDNA from Trametes sp. AH28-2 was expressed in Pichia pastoris, with the highest expression level of 4.0 mg L-1 (1360 U mg-1). The apparent Km (24.6 microM) for ABTS (2,2'-azinobis [3-ethylbenzothia-zoline-6-sulfonic acid]) and the carbohydrate content of the recombinant laccase A (rLacA) are approximately identical to those of the native LacA (nLacA). However, the two enzymes differed in the pH optimum when both ABTS and guaiacol served as substrates. The optimum pH for enzyme stability is 5.5 for rLacA. Thermal stability was also investigated. The mutagenesis of rLacA utilizing low-energy nitrogen ion implantation resulted in the isolation of a yeast clone that produced 7.7 mg L-1 (1085 U mg-1) of laccase, 92.5% more than the nonirradiated control (4.0 mg L-1). Compared with rLacA, the mutant LacA (mLacA) with five amino-acid residue changes in the coding sequence showed a slight change in its catalytic ability but superior thermal stability.

Cloning, Molecular↗
PubMed2005

Use of Ramachandran plot for increasing thermal stability of bacterial formate dehydrogenase.

From analysis of Ramachandran plot for NAD+-dependent formate dehydrogenase from the methylotrophic bacterium Pseudomonas sp. 101 (FDH, EC 1.2.1.2), five amino acid residues with non-optimal values phi and psi have been located in beta- and pi-turns of the FDH polypeptide chain, e.g., Asn136, Ala191, Tyr144, Asn234, and His263. To clarify their role in the enzyme stability, the residues were replaced with Gly by means of site-directed mutagenesis. The His263Gly mutation caused FDH destabilization and a 1.3-fold increase in the monomolecular inactivation rate constant. The replacements Ala191Gly and Asn234Gly had no significant effect on the stability. The mutations Asn136Gly and Tyr144Gly resulted in higher thermal stability and decreased the inactivation rate by 1.2- and 1.4-fold, respectively. The stabilizing effect of the Tyr144Gly mutation was shown to be additive when introduced into the previously obtained mutant FDH with enhanced thermal stability.

Amino Acid Sequence↗
PubMedDate not supplied

Catalytic properties of D-amino acid oxidase in cephalosporin C bioconversion: a comparison between proteins from different sources.

Lacking an efficient process to produce 7-aminocephalosporanic acid from cephalosporin C in a single step, d-amino acid oxidase (DAAO) is of foremost importance in the industrial, two-step process used for this purpose. We report a detailed study on the catalytic properties of the three available DAAOs, namely, a mammalian DAAO and two others from yeast (Rhodotorula gracilis and Trigonopsis variabilis). In comparing the kinetic parameters determined for the three DAAOs, with both cephalosporin C and d-alanine as substrate, the catalytic efficiency of the two enzymes from microorganism is at least 2 orders of magnitude higher than that of pig kidney DAAO. Furthermore, the mammalian enzyme is more sensitive to product inhibition (from hydrogen peroxide and glutaryl-7-aminocephalosporanic acid). Therefore, enzymes from microorganisms appear to be by far more suitable catalysts for bioconversion, although some different minor differences are present between them (e.g., a higher activity of the R. gracilis enzyme when the bioconversion is carried out at saturating oxygen concentration). The mammalian DAAO, even being a poor catalyst, is more stable with respect to temperature than the R. gracilis enzyme in the free form. In any case, for industrial purposes DAAO is used only in the immobilized form where a strong enzyme stabilization occurs.

Animals↗
PubMed1989

New approaches to the preparation and application of firefly luciferase.

Allowing for the lipid nature of firefly luciferase we have developed a new method for obtaining high-activity and high-stability enzyme preparations for bioluminescent microassay. The method includes the step of differential centrifugation in presence of stabilizing additives which entails a partial purification of the enzyme and its essential stabilization likely due to the fact that luciferase retains its lipid environment which plays an important role in catalysis. The resultant luciferase preparation is stable in solution at 4 degrees C for 2-3 months and allows the detection of down to 10(-11) M ATP. A new method has been offered for luciferase immobilization on film carriers precoated with a phospholipid layer. By sorption of the enzyme on such carriers, the samples of immobilized luciferase have been obtained suitable for constructing chemiluminescent biosensors, in the form of luciferase-containing films. There are many-fold applications for detection of ATP micro-quantities.

Animals↗
PubMed1988

Sepiapterin reductase in human amniotic and skin fibroblasts, chorionic villi, and various blood fractions.

Sepiapterin reductase activity has been measured in amniotic fibroblasts by two procedures: one photometric and the other HPLC-fluorimetric. Both can be used for quantitative measurements, but the latter has considerable advantages including smaller standard deviation, much lower detection limit, and less volume of sample required. Sepiapterin reductase activity was also assayed in skin fibroblasts, chorionic villi and various blood fractions including stimulated mononuclear blood cells. Red blood cells have a low specific activity compared to unstimulated mononuclear blood cells, although the latter have a mean value with a high standard deviation. When the mononuclear blood cells were cultured for 5 days, the mean specific activity increased and the range became tighter. Enzyme stability and N-acetylserotonin inhibition were also studied.

Alcohol Oxidoreductases↗
PubMed1990

Synthetic peptide model of an essential region of an aminoacyl-tRNA synthetase.

A 40 amino acid sequence of the unsolved structure of Escherichia coli alanine-tRNA synthetase is essential for tRNA binding and encodes an immunological determinant that cross-reacts with antibodies raised against a eukaryote (insect Bombyx mori) alanine enzyme. The secondary structure of this sequence is predicted to be an amphiphilic alpha-helix that includes one aspartyl and eight glutamyl side chain carboxyl groups. The antibody reactivity and the conformation of a synthetic peptide model of this region (Glu346 to Ser385) were investigated. In addition, double Arg----Gln and Leu----Ala substitutions were separately placed in the enzyme on the hydrophilic and hydrophobic face, respectively, of the predicted helix. These mutations conserve the polar/nonpolar character of each face and retain the potential for helix formation. Circular dichroism spectra of the synthetic peptide model demonstrate the potential for amphiphilic helix formation for the segment from Glu346 to Ser385. The behavior of the mutations in the enzyme, together with earlier data and immunological assays presented here, suggests that one face of the putative helix is an antigenic region of the surface of the enzyme where it contributes to the interaction with alanine tRNA and that the specific sequence of the helix is an important determinant of enzyme stability.

Alanine-tRNA Ligase↗
PubMed1991

Isolation, purification and characterisation of 2-oxoglutarate reductase from Fusobacterium nucleatum.

2-Oxoglutarate reductase from Fusobacterium nucleatum was isolated by thiol-disulphide interchange covalent chromatography. The enzyme was purified approximately 4000-fold and had a molecular mass of 68 kDa. The Michaelis constants for 2-oxoglutarate and NADH were 6.4 x 10(-5) and 0.4 x 10(-5), respectively. The involvement of sulphahydryl groups in catalysis was shown from the inhibition of 2-oxoglutarate reduction in the presence of 2,2'-dipyridyl disulphide and reactivation with 2-mercaptoethanol. Allosteric effectors did not alter the rate of the reaction, or the enzyme stability. With the exception of 2-oxoglutarate, none of the other oxo-acids such as oxaloacetate, pyruvate, 2-oxobutyrate and glyoxylate were reduced. Although 2-oxoglutarate oxidised NADPH to a limited extent (3%), the enzyme was almost entirely specific towards NADH. 2-Oxoglutarate reductase was stable at 45 degrees C for 10 min, while incubation at 60 degrees C abolished all activity.

Alcohol Oxidoreductases↗
PubMed2004

Improving dioxygenase stability by gene chromosome insertion: implementation in immobilized-cell systems.

The immobilization of recombinant cells by using the unstable 3,4-dihydroxyphenylacetate 2,3-dioxygenase was studied as a model. Dioxygenase activity and cell viability were compared in immobilized-cell systems and cells in suspension. Immobilization increased enzyme stability and the efficient degradation of 3,4-dihydroxyphenylacetate. The stability of the cloned enzyme and the viability of the immobilized recombinant cells were well maintained for at least 15 days. We used the strain Escherichia coli CC118-D in which the hpaB gene from Klebsiella pneumoniae, coding for the subunit of 3,4-dihydroxyphenylacetate 2,3-dioxygenase, was inserted into the chromosome. This study has demonstrated that the implementation of E. coli CC118-D in a pilot-scale bioreactor resulted in a 100% stabilization of dioxygenase activity, and could be a useful tool for bioremediation processes.

Bioreactors↗
PubMed2005

Marked decrease in specific activity contributes to disease phenotype in two human glucose 6-phosphate dehydrogenase mutants, G6PD(Union) and G6PD(Andalus).

Clones overexpressing clinical glucose 6-phosphate dehydrogenase (G6PD) mutants Union (c.1360C>T/p.Arg454Cys) and Andalus (c.1361G>A/p.Arg454His), have been constructed. These abolish a salt bridge between Arg454 and Asp 286. One mutant is reportedly a Class II clinical variant and the other a Class I. Kinetic studies of the purified proteins reveal that, for both mutants, kcat is about 10-fold decreased, thus giving a 90% decrease in the WHO assay, and also presumably under physiological conditions. In contrast with unfavourable changes in Vmax for both mutants, Km values for both G6P and NADP+ are decreased approximately 5-fold. Measurements with alternative substrates confirm that G6PD Union, like the wild-type enzyme, follows a rapid-equilibrium random-order mechanism, allowing calculation of enzyme-substrate dissociation constants from initial-rate parameters. The mutations result in several-fold tighter binding of glucose 6-phosphate to the free enzyme. Binding, however, is clearly less productive than with normal enzyme. G6PD mutations are thought to cause haemolytic anaemia by compromising enzyme stability. Both these mutants indeed show somewhat decreased thermostability. However, at 37 degrees C and with NADP+, the stability differences are only moderate. Decreased catalytic efficiency clearly contributes to the disease phenotype of these two mutants, entirely accounting for reported decrease in leukocyte G6PD levels, though not for still lower levels in erythrocytes. Neither the kinetic nor the stability effects appear to justify the different clinical classification of these mutations.

Anemia, Hemolytic↗
PubMed2002

Artificial evolution of an enzyme active site: structural studies of three highly active mutants of Escherichia coli alkaline phosphatase.

The crystal structure of three mutants of Escherichia coli alkaline phosphatase with catalytic activity (k(cat)) enhancement as compare to the wild-type enzyme is described in different states. The biological aspects of this study have been reported elsewhere. The structure of the first mutant, D330N, which is threefold more active than the wild-type enzyme, was determined with phosphate in the active site, or with aluminium fluoride, which mimics the transition state. These structures reveal, in particular, that this first mutation does not alter the active site. The second mutant, D153H-D330N, is 17-fold more active than the wild-type enzyme and activated by magnesium, but its activity drops after few days. The structure of this mutant was solved under four different conditions. The phosphate-free enzyme was studied in an inactivated form with zinc at site M3, or after activation by magnesium. The comparison of these two forms free of phosphate illustrates the mechanism of the magnesium activation of the catalytic serine residue. In the presence of magnesium, the structure was determined with phosphate, or aluminium fluoride. The drop in activity of the mutant D153H-D330N could be explained by the instability of the metal ion at M3. The analysis of this mutant helped in the design of the third mutant, D153G-D330N. This mutant is up to 40-fold more active than the wild-type enzyme, with a restored robustness of the enzyme stability. The structure is presented here with covalently bound phosphate in the active site, representing the first phosphoseryl intermediate of a highly active alkaline phosphatase. This study shows how structural analysis may help to progress in the improvement of an enzyme catalytic activity (k(cat)), and explains the structural events associated with this artificial evolution.

Alkaline Phosphatase↗
PubMed2004

Thermoinactivation analysis of vacuolar H(+)-pyrophosphatase.

Vacuolar H(+)-translocating pyrophosphatase (H(+)-PPase; EC 3.6.1.1) catalyzes both the hydrolysis of PP(i) and the electrogenic translocation of proton from the cytosol to the lumen of the vacuole. Vacuolar H(+)-PPase, purified from etiolated hypocotyls of mung bean (Vigna radiata L.), is a homodimer with a molecular mass of 145 kDa. To investigate the relationship between structure and function of this H(+)-translocating enzyme, thermoinactivation analysis was employed. Thermoinactivation studies suggested that vacuolar H(+)-PPase consists of two distinct states upon heat treatment and exhibited different transition temperatures in the presence and absence of ligands (substrate and inhibitors). Substrate protection of H(+)-PPase stabilizes enzyme structure by increasing activation energy from 54.9 to 70.2 kJ/mol. We believe that the conformation of this enzyme was altered in the presence of substrate to protect against the thermoinactivation. In contrast, the modification of H(+)-PPase by inhibitor (fluorescein 5'-isothiocyanate; FITC) augmented the inactivation by heat treatment. The native, substrate-bound, and FITC-labeled vacuolar H(+)-PPases possess probably distinct conformation and show different modes of susceptibility to thermoinactivation. Our results also indicate that the structure of one subunit of this homodimer exerts long distance effect on the other, suggesting a specific subunit-subunit interaction in vacuolar H(+)-PPase. A working model was proposed to interpret the relationship of the structure and function of vacuolar H(+)-PPase.

Calorimetry, Differential Scanning↗
PubMedDate not supplied

[Role of intramolecular bonds in stability of certain enzymes of the cellulolytic complex].

Stability of C1- and C2-cellulases, CX-exo- and CX-endoglucanases and beta-glucosidase of Aspergillus awamori was studied as affected by monoatomic aliphatic alcohols --methanol, ethanol, propanol and isopropanol; bi- and triatomic alcohols - ethylene glycol and glycerol, urea as well as detergents of dodecyl sulphate and sodium nonilate. The mentioned enzymes are established to manifest the highest activity in 40-60% glycerol. It is also shown that their stability is changed differently under the effect of other alcohols, urea and detergents. The latter testifies to the fact that the studied enzymes are nonidentical, in particular, they differ between themselves by a ratio of intramolecular forces which stabilize their macrostructure.

Alcohols↗
PubMed2006

Effect of organic solvents on the activity and stability of an extracellular protease secreted by the haloalkaliphilic archaeon Natrialba magadii.

The effect of various organic solvents on the activity and stability of an extracellular protease produced by the haloalkaliphilic archaeon Natrialba magadii was tested. This protease was active and stable in aqueous-organic solvent mixtures containing 1.5 M NaCl and glycerol, dimethylsulfoxide (DMSO), N,N-dimethyl formamide, propylenglycol, and dioxane. Among the solvents tested, DMSO, propylenglycol, and glycerol were effective in preserving enzyme stability in suboptimal NaCl concentrations. The stabilizing effect of DMSO on this haloalkaliphilic protease was more efficient at pH 8 than at pH 10, suggesting that DMSO may not substitute for salt to allow halophilic proteins to withstand the effect of high pH values. These results show that Nab. magadii extracellular protease is a solvent tolerant enzyme and suggest a potential application of this haloalkaliphilic protease in aqueous-organic solvent biocatalysis.

Archaeal Proteins↗