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PubMed2002

Characterization of copper/zinc-superoxide dismutase from Pagrus major cDNA and enzyme stability.

A full-length cDNA of 794 bp encoding a putative copper/zinc-superoxide dismutase (Cu/Zn-SOD) from Pagrus major was cloned by the PCR approach. Nucleotide sequence analysis of this cDNA clone revealed that it comprises a complete open reading frame coding for 154 amino acid residues. The deduced amino acid sequence showed high similarity (53-91%) with the sequences of Cu/Zn-SOD from other species. Computer analysis of the residues required for coordinating copper (His-47, 49, 64, and 121) and zinc (His-64, 72, 81, and Asp-84), as well as the two cysteines (58 and 147) that form a single disulfide bond, were well conserved among all reported Cu/Zn-SOD sequences. To further characterize the Pagrus major Cu/Zn-SOD, the coding region was subcloned into an expression vector, pET-20b(+), and transformed into Escherichia coli BL21(DE3). The expression of the Cu/Zn-SOD was confirmed by enzyme activity stained on a native-gel and purified by Ni(2+)-nitrilotriacetic acid Sepharose superflow. Dimer was the major form of the enzyme in equilibrium. The dimerization of the enzyme was inhibited under acidic pH (below 4.0 or higher than 10.0). The half-life was 8.6 min and the inactivation rate constant (k(d)) was 9.69 x 10(-2) min(-1) at 70 degrees C. The enzyme activity was not significantly affected under 4% SDS or 0.5 M imidazole. The enzyme was resistant to proteolysis by both trypsin and chymotrypsin.

Amino Acid Sequence↗
PubMed1995

The structure of Pyrococcus furiosus glutamate dehydrogenase reveals a key role for ion-pair networks in maintaining enzyme stability at extreme temperatures.

BACKGROUND: The hyperthermophile Pyrococcus furiosus is one of the most thermostable organisms known, with an optimum growth temperature of 100 degrees C. The proteins from this organism display extreme thermostability. We have undertaken the structure determination of glutamate dehydrogenase from P. furiosus in order to gain further insights into the relationship between molecular structure and thermal stability. RESULTS: The structure of P. furiosus glutamate dehydrogenase, a homohexameric enzyme, has been determined at 2.2 A resolution and compared with the structure of glutamate dehydrogenase from the mesophile Clostridium symbiosum. CONCLUSIONS: Comparison of the structures of these two enzymes has revealed one major difference: the structure of the hyperthermophilic enzyme contains a striking series of ion-pair networks on the surface of the protein subunits and buried at both interdomain and intersubunit interfaces. We propose that the formation of such extended networks may represent a major stabilizing feature associated with the adaptation of enzymes to extreme temperatures.

Amino Acid Sequence↗
PubMed1998

The crystal structure of Pyrococcus furiosus ornithine carbamoyltransferase reveals a key role for oligomerization in enzyme stability at extremely high temperatures.

The Pyrococcus furiosus (PF) ornithine carbamoyltransferase (OTCase; EC 2.1.3.3) is an extremely heat-stable enzyme that maintains about 50% of its activity after heat treatment for 60 min at 100 degrees C. To understand the molecular basis of thermostability of this enzyme, we have determined its three-dimensional structure at a resolution of 2.7 A and compared it with the previously reported structures of OTCases isolated from mesophilic bacteria. Most OTCases investigated up to now are homotrimeric and devoid of allosteric properties. A striking exception is the catabolic OTCase from Pseudomonas aeruginosa, which is allosterically regulated and built up of four trimers disposed in a tetrahedral manner, an architecture that actually underlies the allostery of the enzyme. We now report that the thermostable PF OTCase (420 kDa) presents the same 23-point group symmetry. The enzyme displays Michaelis-Menten kinetics. A detailed comparison of the two enzymes suggests that, in OTCases, not only allostery but also thermophily was achieved through oligomerization of a trimer as a common catalytic motif. Thermal stabilization of the PF OTCase dodecamer is mainly the result of hydrophobic interfaces between trimers, at positions where allosteric binding sites have been identified in the allosteric enzyme. The present crystallographic analysis of PF OTCase provides a structural illustration that oligomerization can play a major role in extreme thermal stabilization.

Allosteric Site↗
PubMed1975

Temperature-sensitive, osmotic-remedial mutants of Neurospora crassa: osmotic pressure induced alterations of enzyme stability.

Three temperature-sensitive, adenine-requiring mutants of Neurospora crassa were found to be osmotic-remedial when non-penetrating solutes were used to increase the osmolarity of the growth medium. The affected enzyme (adenylosuccinase) from one of the mutants (ad-4, 44206t) was found to have higher levels of activity when the organism was grown at non-permissive temperatures under osmotic-remedial conditions than when it was grown with adenine as a nutritional supplement. The enzyme synthesized at 30 degrees C in the presence of adenine exhibited increased sensitivity to inhibition by high salt concentrations and a lowered stability toward heat denaturation, indicating that the remedial effect may be the result of changes in the physical properties of the enzyme molecule. Temperature shift experiments indicate that the enzyme which is synthesized at permissive temperatures or under osmotic-remedial conditions is also stable in vivo under non-permissive conditions. This suggests that the critical period for temperature sensitivity, and conversely osmotic remediability, may be during protein synthesis or during the conformational folding of the protein.

Adenine↗
PubMed1999

Aromatase inhibitors and enzyme stability.

The effects of two steroidal (4-hydroxyandrostenedione and atamestane) and three non-steroidal (fadrozole, vorozole, and pentrozole) aromatase inhibitors on the levels of aromatase mRNA and protein were examined in vitro and in vivo. Immunocytochemistry revealed increased quantities of immunoreactive aromatase in human choriocarcinoma-derived JEG-3 cells in response to pretreatment with the non-steroidal inhibitors. To elucidate this effect in detail, aromatase protein in JEG-3 cells after treatment with various inhibitors was quantified using an enzyme-linked immunosorbent assay (ELISA). A time-dependent increase in aromatase protein in the cells was observed with all the aromatase inhibitors except 4-hydroxyandrostenedione, whereas aromatase mRNA levels in the cells remained unchanged during the inhibitor treatment. The three non-steroidal agents caused an approximately fourfold increase in aromatase protein in the cells 24 h after the treatment, as compared with untreated controls. The increase in aromatase protein in the cells was not blocked by treatment with cycloheximide, an inhibitor of protein synthesis. The inhibitors also appeared to block the rapid degradation observed in JEG-3 cells after induction by forskolin. In vivo, daily injection of the inhibitors into adult female mice caused increases in levels of both aromatase mRNA and protein in the ovary. The increase in aromatase mRNA in this in vivo study could be explained by an increase in gonadotropin concentrations in response to decreased plasma concentrations of estrogens. In conclusion, we suggest that aromatase inhibitors increase aromatase protein through stabilization and reduced protein turnover.

Animals↗
PubMed1999

Subunit dissociation and inactivation of pyruvate kinase by hydrostatic pressure oxidation of sulfhydryl groups and ligand effects on enzyme stability.

The effect of hydrostatic pressure on the stability of tetrameric rabbit muscle pyruvate kinase was investigated by enzyme activity measurements, size-exclusion chromatography, circular dichroism and fluorescence spectroscopies. Under nonreducing conditions, enzyme activity was irreversibly inhibited by increasing pressure and was completely abolished at 350 MPa. Inhibition was dependent on the concentration of pyruvate kinase, indicating that it was related to pressure-induced subunit dissociation. Size-exclusion chromatography of pressurized samples confirmed a decrease in the proportion of tetramers and an increase in monomers relative to native samples. Addition of dithiothreitol immediately following pressure release led to full recovery of both enzyme activity and of native tetramers. Furthermore, no irreversible inhibition of pyruvate kinase was observed if pressure treatment was carried out in the presence of dithiothreitol. These data suggest that pressure-dissociated monomers undergo conformational changes leading to oxidation of sulfhydryl groups, which prevents correct refolding of native tetramers on decompression. These conformational changes are relatively subtle, as indicated by the lack of significant changes in far-UV circular dichroism and intrinsic fluorescence emission spectra of previously pressurized samples. The effects of various physiological ligands on the pressure stability of pyruvate kinase were also investigated. A slight protection against inhibition was observed in the simultaneous presence of K+, Mg2+ and ADP. Both phosphoenolpyruvate and the allosteric inhibitor, phenylalanine, caused marked stabilization against pressure, suggesting significant energy coupling between binding of these ligands and stabilization of the tetramer.

Animals↗
PubMed1988

Structure-stability relationship in proteins: fundamental tasks and strategy for the development of stabilized enzyme catalysts for biotechnology.

The problem of relationships between the protein structure and its stability comprises two major questions. First, how to elucidate the peculiarities of the protein structure responsible for its stability. Second, knowing the general molecular basis of protein stability, how to change the structure of a given protein in order to increase its stability. This review is an attempt to show the modern state of the first (fundamental) and the second (applied) aspects of the problem.

Bacterial Proteins↗
PubMed1999

Role of Ngamma-acetyldiaminobutyrate as an enzyme stabilizer and an intermediate in the biosynthesis of hydroxyectoine.

Strain CHR63 is a salt-sensitive mutant of the moderately halophilic wild-type strain Halomonas elongata DSM 3043 that is affected in the ectoine synthase gene (ectC). This strain accumulates large amounts of Ngamma-acetyldiaminobutyrate (NADA), the precursor of ectoine (D. Cánovas, C. Vargas, F. Iglesias-Guerra, L. N. Csonka, D. Rhodes, A. Ventosa, and J. J. Nieto, J. Biol. Chem. 272:25794-25801, 1997). Hydroxyectoine, ectoine, and glucosylglycerate were also identified by nuclear magnetic resonance (NMR) as cytoplasmic organic solutes in this mutant. Accumulation of NADA, hydroxyectoine, and ectoine was osmoregulated, whereas the levels of glucosylglycerate decreased at higher salinities. The effect of the growth stage on the accumulation of solutes was also investigated. NADA was purified from strain CHR63 and was shown to protect the thermolabile enzyme rabbit muscle lactate dehydrogenase against thermal inactivation. The stabilizing effect of NADA was greater than the stabilizing effect of ectoine or potassium diaminobutyrate. A (1)H NMR analysis of the solutes accumulated by the wild-type strain and mutants CHR62 (ectA::Tn1732) and CHR63 (ectC::Tn1732) indicated that H. elongata can synthesize hydroxyectoine by two different pathways-directly from ectoine or via an alternative pathway that converts NADA into hydroxyectoine without the involvement of ectoine.

Amino Acids, Diamino↗
PubMed2005

Mutation of regulatory serines of rat tyrosine hydroxylase to glutamate: effects on enzyme stability and activity.

Tyrosine hydroxylase is phosphorylated at four serine residues in its amino-terminus by multiple kinases. Phosphorylation of serine 40 by cAMP-dependent protein kinase results in alleviation of dopamine inhibition [J. Biol. Chem. 267 (1992) 12639]. The other serines are at positions 8, 19, and 31. The effect of phosphorylation at these serines has been investigated using mutated forms of tyrosine hydroxylase containing glutamates at the positions of the serines. The S8E, S19E, and S31E tyrosine hydroxylase variants have similar steady-state kinetic parameters and similar binding affinity for catecholamines to wild-type enzyme. The S8E, S19E, S31E, and S40E variants differ in stability at elevated temperatures. The S40E variant is the least stable, while the others are all more stable than wild-type enzyme. The increased stability of S8E, S19E, and S31E tyrosine hydroxylases may be one of the physiological effects of phosphorylation. It may also have implications for the interpretation of activities of heterogeneous mixtures of tyrosine hydroxylase which have been phosphorylated.

Amino Acid Sequence↗
PubMed2002

Degradation of L-glutamate dehydrogenase from Escherichia coli: allosteric regulation of enzyme stability.

L-glutamate dehydrogenase (GDH) is stable in exponentially growing Escherichia coli cells but is degraded at a rate of 20-30% per hour in cells starved for either nitrogen or carbon. GDH degradation is energy-dependent, and mutations in ATP-dependent proteases, ClpAP or Lon lead to partial stabilization. Degradation is inhibited by chloramphenicol and is completely blocked in relA mutant cells, suggesting that ribosome-mediated signaling may facilitate GDH degradation. Purified GDH has a single tight site for NADPH binding. Binding of NADPH in the absence of other ligands leads to destabilization of the enzyme. NADPH-induced instability and sensitivity to proteolysis is reversed by tri- and dicarboxylic acids or nucleoside di- and triphosphates. GTP and ppGpp bind to GDH at an allosteric site and reverse the destabilizing effects of NADPH. Native GDH is resistant to degradation by several purified ATP-dependent proteases: ClpAP, ClpXP, Lon, and ClpYQ, but denatured GDH is degraded by ClpAP. Our results suggest that, in vivo, GDH is sensitized to proteases by loss of a stabilizing ligand or interaction with an destabilizing metabolite that accumulates in starving cells, and that any of several ATP-dependent proteases degrade the sensitized protein.

ATP-Dependent Proteases↗
PubMed1981

Structure and activity of malate dehydrogenase from the extreme halophilic bacteria of the Dead Sea. 2. Inactivation, dissociation and unfolding at NaCl concentrations below 2 M. Salt, salt concentration and temperature dependence of enzyme stability.

The stability of halophilic malate dehydrogenase increases with increasing salt concentration and with decrease in temperature. Stabilization by various salts, at high salt concentrations, follows the Hofmeister series. The enzyme inactivation rates closely match dissociation of the dimeric enzymes into monomeric subunits and unfolding of the polypeptide chains, as followed by velocity sedimentation, light scattering and circular dichroism measurements. The alpha-helix content goes to zero upon denaturation. Unusual water and salt binding properties of the native enzyme (cf. preceding paper, in this journal) are believed to be largely lost upon enzyme dissociation and unfolding. The properties thus seem to be associated with the intact structure of the enzyme.

Chemical Phenomena↗
PubMed2003

The critical role of the N-terminus of 11beta-hydroxysteroid dehydrogenase type 1, as being encoded by exon 1, for enzyme stabilization and activity.

11beta-Hydroxysteroid dehydrogenase type 1 catalyzes the conversion of cortisone to hormonally active cortisol and has been implicated in the pathogenesis of a number of disorders, including insulin resistance and obesity. Because 11beta-HSD 1 is a membrane protein with a very hydrophobic character, it is difficult to purify it in an active state. Not much is known about the topological and structural determinants of 11beta-HSD 1, although the elucidation of the structure of 11beta-HSD 1 would be a great advantage in identifying specific 11beta-HSD 1 inhibitors. Bacterial expression of full-length or truncated 11beta-HSD 1 forms only led to insoluble proteins or to low amounts of enzyme, not sufficient for crystallization. Recently, we reported that the solubility of 11beta-HSD 1 could be increased by substitution of hydrophobic amino acid residues with arginine without affecting activity. Unfortunately, these truncated and soluble forms of 11beta-HSD 1 exhibited an unstable activity that declined very rapidly. So far, the proteins obtained were not suitable for crystallization. To obtain 11beta-HSD 1 in an active and soluble state, in the present investigation we focused on the amino acid sequence encoded by the first exon. Using bacterial and yeast expression systems, we found that this N-terminal peptide could be divided into two parts that have functions other than to anchor 11beta-HSD 1 into the ER membrane. The first hydrophobic part, consisting of amino acid residues 1-15, represents the membrane spanning domain and anchors 11beta-HSD 1 in the ER membrane. The second hydrophilic part of the peptide, consisting of amino acid residues 16-30, plays a crucial role in stabilizing the catalytic domain of 11beta-HSD 1 and in addition, acts as a spacer to keep the catalytic domain of 11beta-HSD 1 into the lumen of the ER. Evidently, we found that the hydrophilic amino acids 24-30 determine 11beta-HSD 1 enzyme activity. Combined, all information obtained should help to design an optimal 11beta-HSD 1 enzyme in the near future with all desired attributes: soluble, active and easy to obtain and purify in sufficient amounts. This soluble and active 11beta-HSD 1 form should be the basis for our ongoing project, which is the determination of the three dimensional structure of 11beta-HSD 1.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1997

Thiols and neuronal nitric oxide synthase: complex formation, competitive inhibition, and enzyme stabilization.

To elucidate how thiols affect neuronal nitric oxide synthase (nNOS) we studied the binding of thiols to tetrahydrobiopterin (BH4)-free nNOS. Dithiothreitol (DTT), 2-mercaptoethanol, and L- and D-cysteine all bound to the heme with Kd values varying from 0.16 mM for DTT to 41 mM for L-cysteine. DTT, 2-mercaptoethanol, and L-cysteine yielded absorbance spectra with maxima at about 378 and 456 nm, indicative of bisthiolate complexes; the maximum at 426 nm with D-cysteine suggests binding of the neutral thiol. From the results with 2-mercaptoethanol we deduced that in 2-mercaptoethanol-free, BH4-free nNOS the sixth heme ligand is not a thiolate. DTT binding to nNOS containing one BH4 per dimer was biphasic. Apparently, the BH4-free subunit bound DTT with the same affinity as the BH4-free enzyme, whereas the BH4-containing subunit exhibited a > 100-fold lower affinity, indicative of competition between DTT and BH4 binding. Binding of DTT to the BH4-containing subunit was suppressed by L-arginine, whereas high-affinity binding was not affected, suggesting that L-arginine binds only to the BH4-containing subunit. DTT competitively inhibited L-citrulline production by nNOS containing one BH4 per dimer (Ki approximately 11 mM). Comparison of DTT binding and inhibition suggests that the heme of the BH4-free subunit is not involved in catalysis. Thermostability of nNOS was studied by preincubating the enzyme at various temperatures prior to activity determination. At nanomolar concentrations, nNOS was stable at 20 degrees C but rapidly deactivated at higher temperatures (t1/2 approximately 6 min at 37 degrees C). At micromolar concentrations, inactivation was 10 times slower. Absorbance and fluorescence measurements demonstrate that inactivation was not accompanied by major structural changes. The stabilization of nNOS by thiols was illustrated by the fact that omission of 2-mercaptoethanol during preincubation for 10 min at 30 degrees C led to an activity decrease of up to 90%.

Animals↗
PubMed1999

Immunoaffinity layering of enzymes. Stabilization and use in flow injection analysis of glucose and hydrogen peroxide.

A general procedure for the high yield immobilization of enzymes with the help of specific anti-enzyme antibodies is described. Polyclonal antibodies were raised against Aspergillus niger glucose oxidase and horseradish peroxidase in rabbits and the gamma globulin (IgG) fraction from the immune sera isolated by ammonium sulphate fractionation followed by ion-exchange chromatography. Immobilization of glucose oxidase and horseradish peroxidase was achieved by initially binding the enzymes to a Sepharose matrix coupled with IgG isolated from anti-(glucose oxidase) and anti-(horseradish peroxidase) sera, respectively. This was followed by alternate incubation with the IgG and the enzyme to assemble layers of enzyme and antibody on the support. The immunoaffinity-layered preparations obtained thus were highly active and, after six binding cycles, the amount of enzyme immobilized could be raised about 25 times over that bound initially. It was also possible to assemble layers of glucose oxidase using unfractionated antiserum in place of the IgG. The bioaffinity-layered preparations of glucose oxidase and horseradish peroxidase exhibited good enzyme activities and improved resistance to heat-induced inactivation. The sensitivity of a flow injection analysis system for measuring glucose and hydrogen peroxide could be remarkably improved using immunoaffinity-layered glucose oxidase and horseradish peroxidase. For the detection of glucose, a Clark-type oxygen electrode, constructed as a small flow-through cell integrated with a cartridge bearing immunoaffinity-layered glucose oxidase was employed. The hydrogen peroxide concentration was analysed spectrophotometrically using a flow-through cell and the layered horseradish peroxidase packed into a cartridge. The immunoaffinity-layered enzymes could be conveniently solubilized at acid pH and fresh enzyme loaded onto the support. Immunoaffinity-layered glucose oxidase was successfully used for the on-line monitoring of the glucose concentration during the cultivation of Streptomyces cerevisiae.

Animals↗
PubMed1979

The principles of enzyme stabilization. IV. Modification of 'key' functional groups in the tertiary structure of proteins.

The dependence of alpha-chymotrypsin thermostability and catalytic activity on the degree of its amino groups modification has been studied. Modification was carried out by both alkylation (using acrolein with further reduction of Schiff bases by sodium borohydride) and acylation (with siccinic or acetic anhydrides). It has been determined that modification of the majority of titrated amino groups (approximately 80%) only has a slight effect on the first-order rate-constant characterizing the monomolecular process of enzyme thermoinactivation (50 degrees C, pH 8). Thermostability sharply increases (by 120 times) only for a degree of modification higher than 80%, but, nevertheless, the complete substitution of all the titrated amino groups again leads to enzyme destabilization. The conclusion has been drawn that there is only one or two amino groups out out approximately fifteen titrated ones, the modification of which plays a key role in the lateration by the enzyme of its thermostability. The degree of the stabilization effect has been studied relative to both the nature and concentration of the salt added Na2SO4, NaCl, KCl, CCl3COOK, (CH3)4NBr. Ultraviolet absorption (280 nm) of chymotrypsin has also been elucidated with respect to the degree of alkylation of its NH2-groups. The data obtained allowed the conclusion to be drawn that enzyme modification leads to a decrease in the non-electrostatic (hydrophobic) interactions on the surface layer of the globule. As a result, a protein conformation more stable in respect to denaturation (unfolding), is formed.

Acetylation↗
PubMed1981

Proteinases and enzyme stability in crude extracts of castor bean endosperm.

The stability of catalase, fumarase, and isocitrate lyase from deliberately broken organelles in crude extracts from endosperm tissue of castor bean seedlings has been examined. These enzymes are relatively stable at 2 C in extracts from endosperm of 2-day seedlings, but rapid losses of activity occur in extracts from older seedlings. These losses are shown to be brought about by the thiol-proteinase present in the extracts. The inclusion of 35% glycerol prevented the loss of catalase, fumarase, and isocitrate lysase activity, and various inhibitors of proteinases afforded limited protection. The most striking protectant was leupeptin, an inhibitor of serine and thiol-proteinases. Leupeptin completely inhibited the loss of activity of the three enzymes in crude extracts and improved yields when included in the grinding medium.

Journal Article↗
PubMed1983

Asparaginase II of Saccharomyces cerevisiae: comparison of enzyme stability in vivo and in vitro.

Asparaginase II of Saccharomyces cerevisiae is a cell wall mannan containing glycoprotein. Recent studies have demonstrated that asparaginase II activity increases in exponentially growing cell cultures and then decreases as the cells enter the stationary phase. Enzyme inactivation has been attributed to a Zn2+-dependent protease which is synthesized de novo during the late exponential phase [Pauling, K.D., & Jones, G.E. (1980) J. Gen. Microbiol. 117, 423-430; Pauling, K.D., & Jones, G.E. (1980) Biochim. Biophys. Acta 616, 271-282]. We have investigated the mechanism of asparaginase II inactivation using both whole cell suspensions and highly purified enzyme. Our data indicate that the rate of asparaginase II inactivation in cell suspensions is primarily influenced by pH changes that occur as a consequence of cell growth and glucose fermentation and that enzyme inactivation is not dependent on Zn2+ or on de novo protein synthesis. Also, in vitro studies with purified enzyme show kinetics of inactivation that are similar to those observed in vivo. Consequently, involvement of a yeast protease in the inactivation process is relatively unlikely.

Asparaginase↗