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PubMedDate not supplied

beta-Glucosidase and peroxidase stability in crude enzyme extracts from green beans of Vanilla planifolia Andrews.

The extraction method for beta-glucosidase from green vanilla beans has been studied. The effect of storage of green beans and protein extracts on beta-glucosidase and peroxidase activity was investigated: the best method, resulting in the highest enzyme activities, particularly for glucosidase, was through extraction of very fresh green beans in the presence of BisTris propane buffer at pH 8. The best method for storage of the extracts was at -80 degrees C after addition of 15% glycerol, when over 90% of initial activity was still present. Peroxidase activity did not change in frozen beans or in frozen extracts.

Orchidaceae↗
PubMed1994

A conserved glutamic acid bridge in serine carboxypeptidases, belonging to the alpha/beta hydrolase fold, acts as a pH-dependent protein-stabilizing element.

Serine endopeptidases of the chymotrypsin family contain a salt bridge situated centrally within the active site, the acidic component of the salt bridge being adjacent to the catalytically essential serine. Serine carboxypeptidases also contain an acidic residue in this position but it interacts through a short hydrogen bond, probably of low-barrier type, with another acidic residue, hence forming a "glutamic acid bridge." In this study, the residues constituting this structural element in carboxypeptidase Y have been replaced by site-specific mutagenesis. It is demonstrated that the glutamic acid bridge contributes significantly to the stability of the enzyme below pH 6.5 and has an adverse effect at pH 9.5. Carboxypeptidase WII from wheat contains 2 such bridges, and it is more stable than carboxypeptidase Y at acidic pH.

Amino Acid Sequence↗
PubMed1999

NADPH-dependent microsomal electron transfer increases degradation of CYP2E1 by the proteasome complex: role of reactive oxygen species.

Increased levels of cytochrome P450 2E1 (CYP2E1) produced by low-molecular-weight compounds is mostly due to stabilization of the enzyme against proteolytic degradation. CYP2E1, in the absence of substrate or ligand, normally has a short half-life, but the factors which regulate CYP2E1 turnover or trigger its rapid degradation are not known. Since CYP2E1 is active in producing reactive oxygen species, experiments were carried out to evaluate whether reactive oxygen species modulated the degradation of CYP2E1. CYP2E1 present in human liver microsomes was very stable. Addition of the cytosol fraction produced degradation of CYP2E1, and this was enhanced when NADPH was present in the reaction system. Antioxidants or iron chelators which prevent lipid peroxidation, prevented the degradation of CYP2E1 by the cytosolic fraction. Similarly, diphenyleneiodonium chloride, which inhibits NADPH-dependent electron transfer, prevented the degradation of CYP2E1, as did 4-methylpyrazole, a ligand which increases the level of CYP2E1. If microsomes were first incubated with NADPH for 30 min, followed by the addition of these agents, there was no protection against CYP2E1 degradation. Lactacystin, an inhibitor of the proteasome, decreased the degradation of CYP2E1. In intact HepG2 cells transduced to express CYP2E1, proteasome inhibitors elevated steady-state levels of CYP2E1. Steady-state levels of CYP2E1 were increased by about 50% when the cells were incubated with trolox. Trolox decreased the rate of loss of CYP2E1 protein when the cells were treated with cycloheximide. These results suggest that NADPH-dependent production of reactive oxygen species may result in oxidative modification of CYP2E1, followed by rapid degradation of the labilized CYP2E1 by the proteasome complex. It is interesting to speculate that one consequence of the high rates of production of reactive oxygen species by CYP2E1 is its own labilization and subsequent rapid degradation, and this may be a regulatory mechanism to prevent high levels of the enzyme from accumulating within the cell.

Acetylcysteine↗
PubMed1993

Characterization of a mutant alpha-galactosidase gene product for the late-onset cardiac form of Fabry disease.

The expression product of a mutant alpha-galactosidase gene, Q279E (279Gln-->Glu), found in an atypical variant (cardiac form) of Fabry disease, was purified and characterized. It had kinetic properties similar to those of normal alpha-galactosidase, but was markedly thermolabile at neutral pH. Galactose and melibiose at high concentrations stabilized the mutant enzyme in vitro. Its catalytic activity was 15% of that for the normal enzyme, when it was expressed in COS-1 cells at 37 degrees C. The activity increased at 30 degrees C or in the presence of galactose at 37 degrees C. An increase was also observed in lymphoblasts from a patient with this mutation in the presence of galactose or melibiose. We conclude that this mutant protein is posttranslationally inactivated under the neutral conditions in the cells. The possibility of a new therapeutic approach is suggested.

Age of Onset↗
PubMed1998

Amino acid substitutions in the subunit interface enhancing thermostability of Thermoplasma acidophilum citrate synthase.

We have used citrate synthase from Thermoplasma (Tp.) acidophilum as a thermostable model system to investigate the role of hydrophobic interactions in dimer interface for maintaining high temperature stability. Three mutant enzymes were constructed by single amino acid substitutions in the interface helices: Ala97-->Ser, Ala104-->Thr, and Gly209-->Ala. All of the mutations enhanced the thermostability of Tp. citrate synthase, while improving its catalytic properties (Km, Vmax, and specific activity). The highest thermostability was achieved by the Gly209-->Ala substitution. The half-life of irreversible inactivation of the G209A mutant enzyme at 85 degreesC was about 57 min, and the midpoint of guanidinium chloride (GdmCl) induced irreversible denaturation was at 2.0 M GdmCl. Our results showed that amino acid substitutions increasing or decreasing interface hydrophobicity could further increase the thermostability of the Tp. citrate synthase. Thus, interface substitutions affecting the entropy of the unfolded state did not prove to be so critical in protein thermostabilization at higher temperatures.

Chemical Phenomena↗
PubMed2002

An infrared study of the thermal and pH stabilities of the GPI-alkaline phosphatase from bovine intestine.

Alkaline phosphatase (EC 3.1.3.1) from bovine intestine mucosa (BIAP) is a homodimeric metalloenzyme, which hydrolyses nonspecifically phosphate monoesters at alkaline pH with release of inorganic phosphate and alcohol. BIAP is either soluble (sBIAP) or membrane-anchored by a glycosylphosphatidylinositol moiety (GPI-BIAP). This anchor might have some contribution in the stabilization of the GPI-linked protein structure. Our purpose was to study the role of the anchor by using two parameters, the enzymatic activity and the protein conformation, which was analyzed by using FTIR spectroscopy. We determined that the two forms of BIAP show some similarities with the previously described structure of alkaline phosphatase isolated from Escherichia coli and human placenta. Meanwhile GPI-BIAP and sBIAP exhibit similar specific activities, the presence of the anchor increases the thermal and pH stabilities of the enzyme activity and conformation.

Alkaline Phosphatase↗
PubMed1996

Crystallographic investigations of subtilisin BPN' mutants engineered for studying thermal stability.

Subtilisin BPN' (BPN) is an industrially important serine protease that has been extensively investigated in many laboratories. In an effort to improve the thermal stability of the enzyme, researchers at Procter & Gamble have used site-directed mutagenesis techniques to produce several variants of BPN in which residues at the surface of the enzyme have been substituted. We initiated crystallographic studies to determine the structural consequences of these amino acid substitutions. In the course of this work we obtained excellent crystals that correspond to the C2 crystal form of native BPN that has been previously reported. Since the structure reported in that work was of only medium resolution, high-resolution X-ray data for this crystal form of native BPN have been collected and the refinement of the structure has been extended using these new data. Isomorphous crystals of two variants, Q19E and Q271E, have also been grown, high-resolution X-ray data have been collected for these crystals, and the experimental results are described. The structures of the native enzyme and the Q271E variant have been refined and are described.

Binding Sites↗
PubMed1992

In vitro mutagenesis of a xylanase from the extreme thermophile Caldocellum saccharolyticum.

Six mutant xylanases were obtained by in vitro mutagenesis of a xylanase gene from the extremely thermophilic bacterium Caldocellum saccharolyticum. The temperature stability of all enzymes was affected by mutation to various degrees and one of the xylanases had an altered temperature optimum. The mutations had no effect on the pH optimum. The C. saccharolyticum xylanase showed strong homology to several thermophilic and mesophilic xylanases, and comparison of primary sequences allowed the localization of probable active sites and residues involved in thermostability.

Amino Acid Sequence↗
PubMed1995

Physicochemical properties of alkaline serine proteases in alcohol.

The alkaline proteases subtilisin Carlsberg and alcalase possess substantial enzymatic activity even when dissolved in ethanol. The crude enzymes were purified by gel filtration and the main fractions suspended in ethanol to give a translucent suspension. Both the supernatant and the resuspended precipitate after high-speed centrifugation were found to have enzymatic activities. The solubility of subtilisin Carlsberg in anhydrous ethanol was found to be 45.1 micrograms/ml and that of alcalase was 48.1 micrograms/ml by Coomassie blue dye-binding method using bovine serum albumin as a standard. In the presence of water, the solubility of both enzymes increased with water content. The stability of enzymes incubated in ethanol was assayed by their amidase and transesterase activities using Ala-Ala-Pro-Phe-pNA as substrate in phosphate buffer (pH8.2) and Moz-Leu-OBzl as substrate in anhydrous ethanol, respectively. The soluble enzymes have a half-life of about 36 hr and that of suspended enzymes about 50 hr in the amidase activity assay, whereas the same soluble enzymes have a half-life of about several hours and that of suspended enzymes 1 h by the transesterase activity assay. The stability of both enzymes decreased as water concentration increased. The diastereoselectivity of the enzyme-catalyzed hydrolysis of diastereo pairs of tetrapeptide esters, L-Ala-L-Ala-(D- or L-)Pro-L-Phe-OMe and L-Ala-L-Ala-(D- or L-)Ala-L-Phe-OMe, in phosphate is as high as that of the transesterification of these substrates in ethanol. It is concluded that active sites and selectivity of alkaline serine proteases in anhydrous alcohol are probably very similar to those in aqueous solution in spite of the fact that a lower reactivity is usually associated with the enzymes in nonaqueous solvents.

Amino Acid Sequence↗
PubMed2006

Fusion of carbohydrate binding modules from Thermotoga neapolitana with a family 10 xylanase from Bacillus halodurans S7.

Xylanase A of Thermotoga neapolitana contains binding domains both at the N- and C-terminal ends of the catalytic domain. In the N-terminal position it contains two carbohydrate-binding modules (CBM) which belong to family 22. These CBMs bind xylan but not to cellulose. The gene encoding the mature peptide of these CBMs was fused with an alkaline active GH10 xylanase from Bacillus halodurans S7 and expressed in Escherichia coli. The (His)(6) tagged hybrid protein was purified by immobilized metal affinity chromatography and characterized. Xylan binding by the chimeric protein was influenced by NaCl concentration and pH of the binding medium. Binding increased with increasing salt concentration up to 200 mM. Higher extent of binding was observed under acidic conditions. The fusion of the CBM structures enhanced the hydrolytic efficiency of the xylanase against insoluble xylan, but decreased the stability of the enzyme. The optimum temperature and pH for the activity of the xylanase did not change.

Amino Acid Sequence↗
PubMed2006

Intersubunit disulfide interactions play a critical role in maintaining the thermostability of glucose-6-phosphate dehydrogenase from the hyperthermophilic bacterium Aquifex aeolicus.

Proteins from thermophilic microorganisms are stabilized by various mechanisms to preserve their native folded states at higher temperatures. A thermostable glucose-6-phosphate dehydrogenase (tG6PDH) from the hyperthermophilic bacterium Aquifex aeolicus was expressed as a recombinant protein in Escherichia coli. The A. aeolicus G6PDH is a homodimer exhibiting remarkable thermostability (t1/2 = 24 hr at 90 degrees C). Based on homology modeling and upon comparison of its structure with human G6PDH, it was predicted that cysteine 184 of one subunit could form a disulfide bond with cysteine 352 of the other subunit resulting in reinforced intersubunit interactions that hold the dimer together. Site-directed mutagenesis was performed on tG6PDH to convert C184 and C352 to serines. The tG6PDH double mutant exhibited a dramatic decrease in the half-life from 24 hr to 3 hr at 90 degrees C. The same decrease in half-life was also found when either C184 or C352 was mutated to serine. The result indicates that C184 and C352 may play a crucial role in strengthening the dimer interface through disulfide bond formation, thereby contributing to the thermal stability of the enzyme.

Bacteria↗
PubMed1990

Attenuated total reflectance Fourier transform infrared analysis of an acyl-enzyme intermediate of alpha-chymotrypsin.

Attenuated total reflectance Fourier transform infrared (FTIR) spectroscopy was used to obtain signal enhancement of the spectrum of the trans-cinnamoyl-alpha-chymotrypsin acyl-enzyme intermediate. Dilute solutions (as low as 2.5 mg/ml) of enzyme or stabilized acyl-enzyme intermediate were used to form thin films on a germanium crystal surface. The secondary structure of the enzyme thin film was shown to be consistent with the native secondary structure using deconvoluted FTIR data. A novel subtraction technique was used to eliminate interfering spectra of water vapor and protein in critical regions of analysis for esters. This permitted the difference spectra of the one new ester carbonyl bond to be discerned from the 300 or so amide bonds in the protein. The results suggest that the acyl-enzyme exists in two different conformations. This study demonstrates that ir structural information of enzyme-substrate or enzyme-inhibitor complexes can be obtained with dilute protein solutions.

Algorithms↗
PubMed1992

Relationship between the catalytic center and the primary degradation site of triosephosphate isomerase: effects of active site modification and deamidation.

Covalent modification of the active site Glu165 of triosephosphate isomerase (TPI) (EC 5.3.1.1) with the substrate analogue 3-chloroacetol phosphate (CAP) induces conformational changes similar to those observed during catalysis. We have introduced CAP into the active sites of TPI from yeast, chicken, pig, and rabbit, and assessed the effect of this modification on the structural integrity of the protein. CAP binding accelerated the specific deamidation of Asn71 in mammalian TPI. Transverse urea gradient gel electrophoretic analysis showed that the CAP-TPI dimer dissociates more readily than the native dimer. Hybrids composed of one CAP-modified subunit and one native subunit exhibited intermediate stability. The deamidated enzyme was more susceptible to proteases and denaturing conditions. Subtilisin cleaved the rabbit enzyme primarily at the Thr139-Glu140 bond. The resulting peptides remained noncovalently attached, and the enzyme retained catalytic activity. The data provide further evidence of the interactions between the catalytic center and the subunit interface and that the specific deamidation destabilizes the enzyme initiating its degradation. The enhancement of deamidation upon binding of substrate and catalysis suggest that molecular wear and tear may be involved in regulating proteolytic turnover of the enzyme.

Amides↗
PubMed1985

Studies of protein-phospholipid interaction in isolated mitochondrial ubiquinone-cytochrome c reductase.

The interaction between phospholipids, ubiquinone and highly purified ubiquinol-cytochrome c reductase was studied using differential scanning calorimetry. The enzyme complex and its delipidated forms undergo thermodenaturation at 337.3 and 322.7 K, respectively. The reduced reductase is more stable toward thermodenaturation than is the oxidized enzyme. While phospholipids restored enzymatic activity to the delipidated enzyme complex and stabilized the enzyme toward thermodenaturation, ubiquinone showed little effect on the thermostability of ubiquinol-cytochrome c reductase. The effect of phospholipids on the thermotropic properties of ubiquinol-cytochrome c reductase is dependent upon the molecular properties of the phospholipid. When ubiquinol-cytochrome c reductase was embedded in closed asolectin vesicles, an exothermic transition peak was observed upon thermodenaturation. When the asolectin concentration in the reconstituted preparation was less than 0.3 mg/mg protein, an amorphous structure was observed in the electron micrograph and the preparation showed an endothermic transition upon thermodenaturation. The thermotropic properties of the enzyme-phospholipid vesicles were affected by the phospholipid head groups as well as the fatty-acyl chains, with those phospholipids having the most highly unsaturated fatty-acyl chains having the greatest effect. The energy for the exothermic transition may be derived from the collapse, upon thermodenaturation, of a strained interaction between the unsaturated fatty-acyl groups of phospholipids and protein molecules resulting from vesicle formation. The exothermic transition of the enzyme-phospholipid vesicle was abolished when cholesterol was included in the vesicles and when reductase was treated with a proteolytic enzyme prior to incorporation into the phospholipid vesicles.

Animals↗