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At least 505 records · Page 28Linked to original sources
PubMed2000

Substitutions of Asn-726 in the active site of yeast DNA topoisomerase I define novel mechanisms of stabilizing the covalent enzyme-DNA intermediate.

Eukaryotic DNA topoisomerase I (Top1p) catalyzes changes in DNA topology and is the cellular target of camptothecin. Recent reports of enzyme structure highlight the importance of conserved amino acids N-terminal to the active site tyrosine and the involvement of Asn-726 in mediating Top1p sensitivity to camptothecin. To investigate the contribution of this residue to enzyme catalysis, we evaluated the effect of substituting His, Asp, or Ser for Asn-726 on yeast Top1p. Top1N726S and Top1N726D mutant proteins were resistant to camptothecin, although the Ser mutant was distinguished by a lack of detectable changes in activity. Thus, a basic residue immediately N-terminal to the active site tyrosine is required for camptothecin cytotoxicity. However, replacing Asn-726 with Asp or His interfered with distinct aspects of the catalytic cycle, resulting in cell lethality. In contrast to camptothecin, which inhibits enzyme-catalyzed religation of DNA, the His substituent enhanced the rate of DNA scission, whereas the Asp mutation diminished the enzyme binding of DNA. Yet, these effects on enzyme catalysis were not mutually exclusive as the His mutant was hypersensitive to camptothecin. These results suggest distinct mechanisms of poisoning DNA topoisomerase I may be explored in the development of antitumor agents capable of targeting different aspects of the Top1p catalytic cycle.

Amino Acid Substitution↗
PubMed2000

Factors enhancing protein thermostability.

Several sequence and structural factors have been proposed to contribute toward greater stability of thermophilic proteins. Here we present a statistical examination of structural and sequence parameters in representatives of 18 non-redundant families of thermophilic and mesophilic proteins. Our aim was to look for systematic differences among thermophilic and mesophilic proteins across the families. We observe that both thermophilic and mesophilic proteins have similar hydrophobicities, compactness, oligomeric states, polar and non-polar contribution to surface areas, main-chain and side-chain hydrogen bonds. Insertions/deletions and proline substitutions do not show consistent trends between the thermophilic and mesophilic members of the families. On the other hand, salt bridges and side chain-side chain hydrogen bonds increase in the majority of the thermophilic proteins. Additionally, comparisons of the sequences of the thermophile-mesophile homologous protein pairs indicate that Arg and Tyr are significantly more frequent, while Cys and Ser are less frequent in thermophilic proteins. Thermophiles both have a larger fraction of their residues in the alpha-helical conformation, and they avoid Pro in their alpha-helices to a greater extent than the mesophiles. These results indicate that thermostable proteins adapt dual strategies to withstand high temperatures. Our intention has been to explore factors contributing to the stability of proteins from thermophiles with respect to the melting temperatures (T(m)), the best descriptor of thermal stability. Unfortunately, T(m) values are available only for a few proteins in our high resolution dataset. Currently, this limits our ability to examine correlations in a meaningful way.

Animals↗
PubMed1996

Analysis of protein conformational characteristics related to thermostability.

The thermal stability of proteins was studied, 195 single amino acid residue replacements reported elsewhere being analysed for several protein conformational characteristics: type of residue replacement; conservative versus nonconservative substitution; replacement being in a homologous stretch of amino acid residues; change in hydrogen bond, van der Waals and secondary structure propensities; solvent-accessible versus inaccessible replacement; type of secondary structure involved in the substitution; the physico-chemical characteristics to which the thermostability enhancement can be attributed; and the relationship of the replacement site to the folding intermediates of the protein, when known. From the above analyses, some general rules arise which suggest where amino acid substitutions can be made to enhance protein thermostability: substitutions are conservative according to the Dayhoff matrix; mainly occur on conserved stretches of residues; preferentially occur on solvent-accessible residues; maintain or enhance the secondary structure propensity upon substitution; contribute to neutralize the dipole moment of the caps of helices and strands; and tend to increase the number of potential hydrogen bonding or van der Waals contacts or improve hydrophobic packing.

Amino Acid Sequence↗
PubMed2005

In-vitro and in-vivo effects of the CYP2C9*11 polymorphism on warfarin metabolism and dose.

OBJECTIVE: To determine the in-vitro and in-vivo effects of the CYP2C9*11 polymorphism on (S)-warfarin metabolism. METHODS AND RESULTS: The *11 allele that results in mutation of Arg335-->Trp occurred with a frequency of approximately 1% in Caucasian and African-American populations. Four subjects carrying the *1/*11 genotype were identified in a clinical cohort of 192 warfarin patients. Compared to control subjects with the *1/*11 genotype (n=127), the *1/*11 group exhibited a 33% reduction in warfarin maintenance dose, that was independent of study population age or INR. In-vitro studies directed towards understanding the mechanism of reduced in-vivo activity revealed very low levels of holo-CYP2C9.11 expression in insect cells and decreased solubility in the presence of detergent. Membrane preparations of CYP2C9.11 contained inactive P420 and exhibited a shorter half-life for thermally induced conversion of P450 to P420 than CYP2C9.1. Metabolic studies demonstrated that functional CYP2C9.11 possessed similar (S)-warfarin hydroxylation regioselectivity and modestly reduced catalytic efficiency relative to the wild-type enzyme. CONCLUSIONS: In-vivo reduction in CYP2C9 (S)-warfarin activity due to the CYP2C9*11 polymorphism may largely be a consequence of decreased enzyme stability resulting in compromised expression of holo-enzyme. Increased enzyme lability of CYP2C9.11 may be related to improper folding due to the disruption of conserved salt-bridge and hydrogen bonding contacts in the loop region between the J and J' helices of the protein.

Anticoagulants↗
PubMed2000

Thermostabilization of proteins by diglycerol phosphate, a new compatible solute from the hyperthermophile Archaeoglobus fulgidus.

Diglycerol phosphate accumulates under salt stress in the archaeon Archaeoglobus fulgidus (L. O. Martins, R. Huber, H. Huber, K. O. Stetter, M. S. da Costa, and H. Santos, Appl. Environ. Microbiol. 63:896-902, 1997). This solute was purified after extraction from the cell biomass. In addition, the optically active and the optically inactive (racemic) forms of the compound were synthesized, and the ability of the solute to act as a protecting agent against heating was tested on several proteins derived from mesophilic or hyperthermophilic sources. Diglycerol phosphate exerted a considerable stabilizing effect against heat inactivation of rabbit muscle lactate dehydrogenase, baker's yeast alcohol dehydrogenase, and Thermococcus litoralis glutamate dehydrogenase. Highly homologous and structurally well-characterized rubredoxins from Desulfovibrio gigas, Desulfovibrio desulfuricans (ATCC 27774), and Clostridium pasteurianum were also examined for their thermal stabilities in the presence or absence of diglycerol phosphate, glycerol, and inorganic phosphate. These proteins showed different intrinsic thermostabilities, with half-lives in the range of 30 to 100 min. Diglycerol phosphate exerted a strong protecting effect, with approximately a fourfold increase in the half-lives for the loss of the visible spectra of D. gigas and C. pasteurianum rubredoxins. In contrast, the stability of D. desulfuricans rubredoxin was not affected. These different behaviors are discussed in the light of the known structural features of rubredoxins. The data show that diglycerol phosphate is a potentially useful protein stabilizer in biotechnological applications.

Alcohol Dehydrogenase↗
PubMed1988

Genetic studies of protein stability and mechanisms of folding.

In the past few years, analysis of mutationally altered proteins has joined more traditional biophysical methods as an important experimental approach to the study of protein folding. Single amino acid substitutions have been found to reduce the stability of the native state by as much as 3 kcal mol-1, a significant fraction of the marginal stability of the folded conformation, and to significantly alter the rates of folding or unfolding. Mutations can also significantly affect the ability of a protein to fold in vivo. However, at present there is great uncertainty in accounting for or predicting the effects of mutations on stability and folding, even qualitatively. Future studies of mutant proteins are likely to provide further insights into the roles of individual residues and interactions in determining the three-dimensional structures of proteins. For the full potential of this approach to be realized, however, many mutant proteins will have to be systematically studied using structural, thermodynamic, and kinetic methods. The results of these experiments may aid in developing and refining improved theoretical treatments of the energetics of protein conformation.

Drug Stability↗
PubMed2002

Stability of cytochrome P450 enzymes in human liver samples stored in different tissue preservation buffers.

The effects of various tissue preservation buffers on cytochrome P450 (P450)-mediated activities of microsomes prepared from fresh surgical liver samples were examined. Two individual human liver samples were obtained, and three portions of each were preserved in one of three solutions: phosphate buffer, Krebs-Heinseleit solution, or University of Wisconsin solution. Microsomes were prepared within 24 h, 3 days, and 7 days after the resection of the samples. Marker P450 activities were measured. Liver microsomes prepared within 24 h displayed similar ethoxyresorufin O-deethylation, bufuralol 1'-hydroxylation (BF 1'-OH), and chlorzoxazone 6-hydroxylation activities in all preservation solutions, whereas S-mephenytoin 4'-hydroxylation and midazolam 1'-hydroxylation activities displayed some variation depending on the preservation buffer used. Most of the marker P450 activities were stable for 3 days after removal from surgical patients and declined at 7 days; however, a decline in BF-1'OH activity was observed even at day 3. These results suggest P450-specific activity depends on the method by which human liver samples are preserved. Moreover, the results of these studies establish the minimum tissue preservation criteria that, when met, qualify the drug disposition data generated from subcellular fractions derived from a particular surgical tissue sample.

Buffers↗
PubMed1988

Periplasmic nonspecific acid phosphatase II from Salmonella typhimurium LT2. Crystallization, detergent reactivation, and phosphotransferase activity.

The periplasmic nonspecific acid phosphatase II from Salmonella typhimurium was purified to homogeneity from a mutant strain that overproduces the enzyme (Uerkvitz, W., and Beck, C.F. (1981) J. Biol. Chem. 256, 382-389). It was shown that the enzyme transfers phosphate groups from organic phosphoric acid esters (donors) to water as well as to the 2'-, 3'-, or 5'-hydroxyls of nucleosides, nucleotides, and other compounds with free hydroxyl groups (acceptors). The enzyme was crystallized in two forms by precipitation with polyethylene glycol. Needles were formed in buffer containing Mg2+, whereas thin rectangular plates appeared in the presence of the non-ionic detergent n-octyl glucoside. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under partially or completely denaturating conditions revealed that the native enzyme is a tetramer consisting of identical 24-kDa monomers. Owing to surface inactivation, polyethylene glycol, non-ionic, or Zwitterionic detergents are indispensable for enzyme stability. The detergents are able to reactivate inactivated enzyme when present near or above their critical micelle concentration.

Acid Phosphatase↗
PubMed1975

The mechanism of action of glycosidases.

The factors that may contribute to the rate enhancement observed with enzymatic versus non-enzymatic hydrolysis of glycosides are discussed. The nature of the active site as deduced from labelling studies with beta-glucosidases is described. A two-step mechanism involving either an enzyme stabilized glycosyl ion or a covalent glycosyl-enzyme intermediate is proposed. Experiments with a beta-glucosidase from almonds show that even with 2-deoxy glucosides with good leaving groups as aglycon which are hydrolyzed 1000 times more slowly than the corresponding glucosides, the deglucosylation step is faster than the cleavage of the glycosidic bond.

Aspergillus↗
PubMed1984

Glutathione disulfide inactivates, destabilizes, and enhances proteolytic susceptibility of fructose-1,6-bisphosphate aldolase.

Disulfides (glutathione disulfide, cystine, cystamine) caused a first-order inactivation of rabbit-muscle fructose-1,6-bisphosphate aldolase at pH values of 7.4 and above. Inactivation by glutathione disulfide was partially reversed by reducing agents, but the enzyme became irreversibly inactivated with time. The disulfide-inactivated aldolase had a lower transition temperature and enthalpy of denaturation than the native enzyme. In addition, the disulfide-inactivated enzyme was extensively degraded by proteinases, whereas the native enzyme was resistant. Mixed disulfides were formed; a maximum ratio of 4-5 mol of glutathione/mol of the aldolase tetramer was found. The number of titratable--SH groups on aldolase decreased by 16 (out of 32 total on the control enzyme) after inactivation by glutathione disulfide, indicating that other oxidation reactions in addition to those resulting in mixed disulfides occurred. The substrate, fructose 1,6-bisphosphate, prevented inactivation of aldolase by glutathione disulfide, the formation of glutathione-enzyme mixed disulfides, thermodynamic destabilization of the enzyme, and a decrease of--SH groups on the enzyme. These data indicate that covalent modification of aldolase by biological disulfides is important in modulating enzyme stability and vulnerability to proteinases as well as enzyme activity and that the substrate protects against modification by disulfides.

Animals↗
PubMed2006

Protein stability and the evolution of the cell membrane.

Cholesterol has been shown to regulate the activity of several membrane proteins. Although this phenomenon represents an important factor in the regulation of ion homeostasis, insights are needed to fully understand the role of this lipid in cell function in order to better comprehend the effect of bilayer components upon membrane function. Since evolution has shaped the composition of the membrane bilayer, it becomes of interest to study these changes in parallel with the many functions of membranes such as ion transport. The present study employing a plasma membrane preparation obtained from calf ventricular muscle demonstrates that cholesterol partially inhibits the Ca(2+),Mg(2+)-ATPase as the catalytic function of the calcium pump, when incubation reaction temperatures are below 42 degrees C. In contrast, when incubation reaction temperatures are above 42 degrees C, cholesterol apparently promotes enzyme stabilization reflected in higher activity. Although the activation energy values for the enzyme are almost the same at ranges between 15 and 40 degrees C, the use of elevated temperatures promote higher enzyme inactivation rates in control than in cholesterol enriched membranes. Cholesterol apparently is promoting stabilization that in turn protects the enzyme against thermal inactivation. This protective effect is reflected in a decrease of inactivation rate values and energy released during enzyme catalysis. The modification of many membrane properties throughout million of years made it possible for new evolutionary driving forces to show themselves as new characteristics in eukaryotes such as the one discussed in this study, dealing with the presence of cholesterol in the cell membrane directly associated to the promotion of protein thermostability.

Animals↗
PubMed2003

Comparison of family 12 glycoside hydrolases and recruited substitutions important for thermal stability.

As part of a program to discover improved glycoside hydrolase family 12 (GH 12) endoglucanases, we have studied the biochemical diversity of several GH 12 homologs. The H. schweinitzii Cel12A enzyme differs from the T. reesei Cel12A enzyme by only 14 amino acids (93% sequence identity), but is much less thermally stable. The bacterial Cel12A enzyme from S. sp. 11AG8 shares only 28% sequence identity to the T. reesei enzyme, and is much more thermally stable. Each of the 14 sequence differences from H. schweinitzii Cel12A were introduced in T. reesei Cel12A to determine the effect of these amino acid substitutions on enzyme stability. Several of the T. reesei Cel12A variants were found to have increased stability, and the differences in apparent midpoint of thermal denaturation (T(m)) ranged from a 2.5 degrees C increase to a 4.0 degrees C decrease. The least stable recruitment from H. schweinitzii Cel12A was A35S. Consequently, the A35V substitution was recruited from the more stable S. sp. 11AG8 Cel12A and this T. reesei Cel12A variant was found to have a T(m) 7.7 degrees C higher than wild type. Thus, the buried residue at position 35 was shown to be of critical importance for thermal stability in this structural family. There was a ninefold range in the specific activities of the Cel12 homologs on o-NPC. The most and least stable T. reesei Cel12A variants, A35V and A35S, respectively, were fully active. Because of their thermal tolerance, S. sp. 11AG8 Cel12A and T. reesei Cel12A variant A35V showed a continual increase in activity over the temperature range of 25 degrees C to 60 degrees C, whereas the less stable enzymes T. reesei Cel12A wild type and the destabilized A35S variant, and H. schweinitzii Cel12A showed a decrease in activity at the highest temperatures. The crystal structures of the H. schweinitzii, S. sp. 11AG8, and T. reesei A35V Cel12A enzymes have been determined and compared with the wild-type T. reesei Cel12A enzyme. All of the structures have similar Calpha traces, but provide detailed insight into the nature of the stability differences. These results are an example of the power of homolog recruitment as a method for identifying residues important for stability.

Amino Acid Sequence↗
PubMed1995

Effects of two mutations detected in medium chain acyl-CoA dehydrogenase (MCAD)-deficient patients on folding, oligomer assembly, and stability of MCAD enzyme.

We have used expression of human medium chain acyl-CoA dehydrogenase (MCAD) in Escherichia coli as a model system for dissecting the molecular effects of two mutations detected in patients with MCAD deficiency. We demonstrate that the R28C mutation predominantly affects polypeptide folding. The amounts of active R28C mutant enzyme produced could be modulated between undetectable to 100% of the wild-type control by manipulating the level of available chaperonins and the growth temperature. For the prevalent K304E mutation, however, the amounts of active mutant enzyme could be modulated only in a range from undetectable to approximately 50% of the wild-type, and the assembled mutant enzyme displayed a decreased thermal stability. Two artificially constructed mutants (K304Q and K304E/D346K) yielded clearly higher amounts of active MCAD enzyme than the K304E mutant but were also responsive to chaperonin co-overexpression and growth at low temperature. The thermal stability profile of the K304E/D346K double mutant was shifted to even lower temperatures than that of the K304E mutant, whereas that of the K304Q mutant was closely similar to the wild-type. Taken together, the results show that the K304E mutation affects (i) polypeptide folding due to elimination of the positively charged lysine and (ii) oligomer assembly and stability due to replacement of lysine 304 with the negatively charged glutamic acid.

Acyl-CoA Dehydrogenase↗
PubMed1995

High-pressure stabilization of alpha-chymotrypsin entrapped in reversed micelles of aerosol OT in octane against thermal inactivation.

alpha-Chymotrypsin (CT) solubilized in reversed micelles of sodium bis-(2-ethylhexyl)-sulfosuccinate (AOT) undergoes thermal inactivation and the enzyme stability decreases significantly when temperature increases (25-40 degrees C). The half-life of CT in micelles shows a bell-shaped dependence on the degree of hydration of AOT (wo) analogous to the previously obtained dependence on wo for the enzyme activity. The optima of catalytic activity and thermal stability have been observed under conditions where the diameter of the inner aqueous cavity of the micelle is close to the size of the enzyme molecule (wo = 10). Application of high hydrostatic pressure in the range of 1-1500 atm (bar) stabilizes CT against thermal inactivation at all hydration degrees (wo) from 7 to 20; the stabilization effect is most pronounced under the experimental conditions being far from the optimum for catalytic activity.

Animals↗
PubMed2006

Mechanistic implications of the structure of the mixed-disulfide intermediate of the disulfide oxidoreductase, 2-ketopropyl-coenzyme M oxidoreductase/carboxylase.

The structure of the mixed, enzyme-cofactor disulfide intermediate of ketopropyl-coenzyme M oxidoreductase/carboxylase has been determined by X-ray diffraction methods. Ketopropyl-coenzyme M oxidoreductase/carboxylase belongs to a family of pyridine nucleotide-containing flavin-dependent disulfide oxidoreductases, which couple the transfer of hydride derived from the NADPH to the reduction of protein cysteine disulfide. Ketopropyl-coenzyme M oxidoreductase/carboxylase, a unique member of this enzyme class, catalyzes thioether bond cleavage of the substrate, 2-ketopropyl-coenzyme M, and carboxylation of what is thought to be an enzyme-stabilized enolacetone intermediate. The mixed disulfide of 2-ketopropyl-coenzyme M oxidoreductase/carboxylase was captured through crystallization of the enzyme with the physiological products of the reaction, acetoacetate, coenzyme M, and NADP, and reduction of the crystals with dithiothreitol just prior to data collection. Density in the active-site environment consistent with acetone, the product of reductive decarboxylation of acetoacetate, was revealed in this structure in addition to a well-defined hydrophobic pocket or channel that could be involved in the access for carbon dioxide. The analysis of this structure and that of a coenzyme-M-bound form provides insights into the stabilization of intermediates, substrate carboxylation, and product release.

Acetoacetates↗
PubMed2002

The improvement of lipase secretion and stability by addition of inert compounds into Acinetobacter calcoaceticus cultures.

Acinetobacter calcoaceticus BD413 produces variable amounts of an exocellular lipase that becomes rapidly inactivated upon secretion. To achieve high yield and protect the enzyme, we assayed the addition of several inert compounds to cell-free supernatants, cell fractions, and whole cultures. Glass beads, poly(ethylene glycol) 600, Triton X-100, saccharose, gum arabic, and beta-cyclodextrin were among the compounds tested. beta-Cyclodextrin and gum arabic (and saccharose to a lesser extent) were effective enzyme stabilizers in cell-free supernatants, while gum arabic, glass beads, and Triton X-100 improved lipase secretion from cells, and, therefore, total lipase yield (30-50%, according to the additive). In whole cultures, beta-cyclodextrin was the most effective additive, particularly in combination with glass beads or gum arabic. Indeed, cultures containing beta-cyclodextrin plus gum arabic were able to maintain 95% (+/- 1.5%) of the initial lipase activity for more than 16 h, while control cultures with no additives maintained only 10% (+/- 4%) of the enzyme activity after the same period. In conclusion, the addition of inert compounds in cultures may be considered a useful approach for achieving increased yield and lipase stabilization, amenable for downstream processing.

Acinetobacter calcoaceticus↗
PubMed2006

Site-directed mutagenesis of a yeast gene for improvement of enzyme thermostability.

Enzyme stability is one of the critical factors to construct an efficient biological conversion system. Xylitol dehydrogenase (XDH) from Pichia stipitis is one of the key enzymes for bio-ethanol fermentation system from xylose. Previously, we tried to improve thermostability of XDH by introduction of structural zinc into the enzyme and successfully obtained a mutant, named C4 mutant, with an increased unfolding temperature (J. Biol. Chem., 280:10340-10349, 2005). We focused on further improvement of the thermostability of XDH in this study and employed subsequent site directed mutagenesis in structural zinc binding region for stabilizing the structural zinc binding loop. Two variants (C4/F98R and C4/E101F) showed higher thermostability than C4 mutant judged by thermal inactivation of enzyme activity and thermal transition temperature.

Binding Sites↗
PubMed1989

Structure-function correlations of polyamine analog-induced increases in spermidine/spermine acetyltransferase activity.

The cytosolic enzyme, spermidine/spermine acetyltransferase (SSAT), is distinguished by its role in polyamine interconversion and by its high inducibility in response to a variety of physiological and pharmacological stimuli. Among a series of fifteen polyamines and polyamine analogs, the most potent inducers of SSAT activity in cultured L1210 cells were found to be N1,N8-bis(ethyl)spermidine (BES) and N1,N12-bis(ethyl)spermine (BESm). Over a 24-hr exposure at 10 microM, enzyme activity rose 13- and 16-fold with BES and BESm, respectively, compared to 2- to 3-fold with the anticancer agent, methylglyoxal bis(guanylhydrazone). The increase in enzyme activity by BESm began rapidly and continued steadily with time so that by 48 hr it increased to about twenty times control. By inhibitor studies, the increase was found to be due to elevated protein synthesis predominantly at the level of translation and to an apparent prolongation of enzyme half-life related to enzyme stabilization. Among the analogs, the structural requirements for maximum enzyme induction were found to be critically dependent on aminopropyl moieties and on the presence, size and location of the alkyl groups. By structure-function comparisons, it was deduced that the known abilities of BES and BESm to regulate ornithine and S-adenosylmethionine decarboxylase activities or to inhibit cell growth occur independently of their effects on SSAT activity in L1210 cells.

Acetyltransferases↗