Effects of amino acid replacements around the reactive site of chicken ovomucoid domain 3 on the inhibitory activity toward chymotrypsin and trypsin

We have previously shown that replacing the P1-site residue(Ala) of chicken ovomucoid domain 3 (OMCHI3) with a Met or Lysresults in the acquisition of inhibitory activity toward chymotrypsinor trypsin, respectively. However, the inhibitory activitiesthus induced are not strong. In the present study, we introducedadditional amino acid replacements around the reactive siteto try to make the P1-site mutants more effective inhibitorsof chymotrypsin or trypsin. The amino acid replacement AspTyrat the P2' site of OMCHI3(P1Met) resulted in conversion to a35000-fold more effective inhibitor of chymotrypsin with aninhibitor constant (K_i) of 1.17x10^–11 M. The K_i valueof OMCHI3(P1Met, P2'Ala) indicated that the effect on the interactionwith chymotrypsin of removing a negative charge from the P2'site was greater than that of introducing an aromatic ring.Similarly, enhanced inhibition of trypsin was observed whenthe AspTyr replacement was introduced into the P2' site of OMCHI3(P1Lys).Two additional replacements, AspAla at the P4 site and ArgAlaat the P3' site, made the mutant a more effective inhibitorof trypsin with a K_i value of 1.44x10^–9 M. By contrast,ArgAla replacement at the P3' site of OMCHI3(P1Met, P2'Tyr)resulted in a greatly reduced inhibition of chymotrypsin, andAspAla replacement at the P4 site produced only a small changewhen compared with a natural variant of OMCHI3. These resultsclearly indicate that not only the P1-site residue but alsothe characteristics, particularly the electrostatic properties,of the amino acid residues around the reactive site of the proteaseinhibitor determine the strength of its interactions with proteases.Furthermore, amino acids with different characteristics arerequired around the reactive site for strong inhibition of chymotrypsinand trypsin.     

Engineering subtilisin YaB: restriction of substrate specificity by the substitution of Gly124 and Gly151 with Ala

The 3-D structure of subtilisin YaB was computer modelled using thestructures of subtilisin BPN', subtilisin Carlsberg and thermitase astemplates. Gly124 and Gly151 located on both sides of the waist of the S1pocket were selected for site-directed mutagenesis based on the modelledstructure. The mutated ale genes coding for the mutant subtilisin YaB wereexpressed in Bacillus subtilis DB104. All of the G124 and G151 series ofmutants exhibited an increase of relative catalytic activity forelastin-orcein against casein and myofibrillar proteins. The S1 substratespecificity of G124A, G124V and G151A mutants were assessed using variouscarbobenzoxy-amino acid-nitrophenyl esters and succinyl-Ala-Ala-(Pro orVal)-(Ala, Phe or Leu)-p- nitroanilide [AA(P/V) (A/F/L)]. While G124A andG124V mutants hydrolyzed only Ala and Gly esters, G151A mutant hydrolyzedAla, Leu and Gly esters. The G124A and G124V mutants did not hydrolyze AAPFand AAPL. However, these two mutants hydrolyzed AAPA and AAVA with kcat/Kmvalues approximately 3-10-fold higher than those of the wild-type enzyme.The G151A mutant did not hydrolyze AAPF, but hydrolyzed AAPL, AAPA and AAVAwith kcat/Km values approximately 1-4-fold higher than those of thewild-type enzyme. These results clearly indicate that the S1 substratespecificity of G124A and G124V mutants was restricted to Ala and Gly, andG151A mutant to Ala, Gly and Leu.     

Trp59 to Tyr substitution enhances the catalytic activity of RNase T1 and of the Tyr to Trp variants in positions 24, 42 and 45

Using point mutated overproducing strains of E.coli, ribonucleaseT1 was prepared with the single substitutions Tyr24Trp, Tyr42Trp,Tyr45Trp or Trp59Tyr and the corresponding double substitutionsTyr24Trp/Trp59Tyr, Tyr42Trp/Trp59Tyr and Tyr45Trp/Trp59Tyr.Steady state kinetics of the transesterification reaction forthe two dinucleoside monophosphate substrates guanylyl-3', 5'-cytidineand guanylyl-3', 5'-adenosine indicate that the tryptophan canbe introduced in different positions within the ribonucleaseT1 molecule without abolishing enzymatic activity. The Trp59Tyrexchange even enhances catalysis of the cleavage reaction (k_cat/K_m)relative to the wild type enzyme and similar effects are foundwith single tyrosine to tryptophan substitutions. For the pHdependencies of the guanylyl-3', 5'-cytidine transesterificationreaction of wild type ribonuclease T1 and of the variants, typicallybell-shaped curves are observed with a plateau in the rangepH 4.5–7.0. Their shapes and slopes indicate that theenzymes are comparable in their macroscopic pK_a, values. AtpH 7.5, the variant Tyr45Trp/Trp59Tyr shows a more than 3-foldhigher transesterification activity for guanylyl-3', 5'-adenosineand a 2-fold increase for guanylyl-3', 5'-cytidine comparedto the wild type enzyme, i.e. this variant catalyses the transesterificationof the substrate guanylyl-3', 5'-adenosine with the same orbetter efficiency as guanylyl-3', 5'-cytidine.     

The specificity and the ability of Aspergillus feruloyl esterase to release p‐coumaric acid from complex cell walls of oat hulls

p‐Coumaric acid (4‐hydroxycinnamic) and ferulic acid (4‐hydroxy‐3‐methoxycinnamic), two major hydroxycinnamic acids found in the complex cell walls of oat hulls, act as cross‐linking agents between lignin and polysaccharides or between polysaccharides. As such, they are inhibitory to the biodegradation of cell walls by microorganisms. A previous study showed that Aspergillus feruloyl esterase with Trichoderma xylanase was able to break the ester linkage between ferulic acid and the attached sugar, releasing ferulic acid from the cell wall. The objective of this study was to investigate the specificity and the ability of Aspergillus feruloyl esterase to release p‐coumaric acid from oat hulls. The results show there was no extensive release of p‐coumaric acid in both the absence and presence of Trichoderma xylanase by Aspergillus feruloyl esterase. This indicates a specificity of Aspergillus feruloyl esterase, which is more active only on esters of certain hydroxycinnamic acids; in this case, Aspergillus feruloyl esterase will only sufficiently break the ester‐linked feruloyl group but not the p‐coumaroyl group in the complex cell walls of oat hulls. Copyright © 2004 Society of Chemical Industry     

Study of the changes in the structure and optical properties of MgxCo1-xCr2-yAlyO4 spinel caused by A/B site ion substitution

In this work, nanosubmicron blue-green pigment powder based on the composition of Mg_xCo_1-xCr_2-yAl_yO₄（0 = x ≤ 1, 0 = y ≤ 2）was prepared by a gel casting method. X-ray diffraction (XRD), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), Rietveld refinement with GSAS (General Structure Analysis System), and UV–Vis absorption spectroscopy were used to study the phase composition, grain size, morphology, cation distribution in the crystal structure and spectral absorption of the samples. Colour parameters were also studied by using a colour measurement spectrophotometer. The studies demonstrate that the distribution of cations in the crystal structure is disordered and that divalent and trivalent cations are mixed to occupy tetrahedral and octahedral sites. Furthermore, the substitution of ions at the A/B site leads to a change in the cation distribution ratio at the tetrahedral and octahedral sites. With increasing Mg²⁺ doping concentration, the inversion parameter of the spinel increases, while with increasing Al³⁺ doping concentration, the inversion parameter of the spinel decreases. In addition, changes in the calcining atmosphere lead to a change in the oxygen vacancy content in the structure. Under the condition of a reductive atmosphere, the oxygen vacancy content significantly increases, and the inversion parameter also increases. The colour difference for the synthesized Mg_xCo_1-xCr_2-yAl_yO₄ spinel powder is related to the proportion of chromophore ions occupying tetrahedral and octahedral sites and the number of oxygen vacancies.     

Lipid specificity and location of the sterol carrier protein-2 fatty acid-binding site: A fluorescence displacement and energy transfer study

Although it was recently recognized that sterol carrier protein-2 (SCP-2) interacts with fatty acids, little is known regarding the specificity of SCP-2 for long-chain fatty acids or branched-chain fatty-acid-like molecules. Likewise the location of the fatty-acid binding site within SCP-2 is unresolved. A fluorescent cis-parinaric acid displacement assay was used to show that SCP-2 optimally interacted with 14–22 carbon chain lipidic molecules: polyunsaturated fatty acids > monounsaturated, saturated > branched-chain isoprenoids > branched-chain phytol-derived fatty acids. In contrast, the other major fatty-acid binding protein in liver, fatty-acid binding protein (L-FABP), displayed a much narrower carbon chain preference in general: polyunsaturated fatty acids > branched-chain phytol-derived fatty acids > 14- and 16-carbon saturated > branched-chain isoprenoids. However, both SCP-2 and l-FABP displayed a very similar unsaturated fatty-acid specificity profile. The presence and location of the SCP-2 lipid binding site were investigated by fluorescence energy transfer. The distance between the SCP-2 Trp⁵⁰ and bound cis-parinaric acid was determined to be 40 Å. Thus, the SCP-2 fatty-acid binding site appeared to be located on the opposite side of the SCP-2 Trp⁵⁰. These findings not only contribute to our understanding of the SCP-2 ligand binding site but also provide evidence suggesting a potential role for SCP-2 and/or L-FABP in metabolism of branched-chain fatty acids and isoprenoids.     

Trail-following pheromones of the rhinotermitidae: Approaches to their authentication and specificity

A method for determining the authenticity of subterranean termite trail pheromones is suggested and utilized to verify the presence of trail pheromones inReticulitermes virginicus, R. flavipes, andR. tibialis. In addition, a possible trail pheromone has been demonstrated forCoptotermes formosanus. A choice bioassay method shows that the above trail pheromones are species specific.     

Structural and functional analysis of a truncated form of Saccharomyces cerevisiae ATP sulfurylase: C-terminal domain essential for oligomer formation but not for activity

ATP sulfurylase catalyzes the first step in the activation ofsulfate by transferring the adenylyl-moiety (AMP) of ATP tosulfate to form adenosine 5'-phosphosulfate (APS) and pyrophosphate(PP_i). Subsequently, APS kinase mediates transfer of the -phosphorylgroup of ATP to APS to form 3'-phosphoadenosine 5'-phosphosulfate(PAPS) and ADP. The recently determined crystal structure ofyeast ATP sulfurylase suggests that its C-terminal domain isstructurally quite independent from the other domains, and notessential for catalytic activity. It seems, however, to dictatethe oligomerization state of the protein. Here we show thattruncation of this domain results in a monomeric enzyme withslightly enhanced catalytic efficiency. Structural alignmentof the C-terminal domain indicated that it is extremely similarin its fold to APS kinase although not catalytically competent.While carrying out these structural and functional studies asurface groove was noted. Careful inspection and modeling revealedthat the groove is sufficiently deep and wide, as well as properlypositioned, to act as a substrate channel between the ATP sulfurylaseand APS kinase-like domains of the enzyme. Received July 17, 2003; revised October 27, 2003; accepted October 28, 2003     

{alpha}-Amylase from Bacillus licheniformis mutants near to the catalytic site: effects on hydrolytic and transglycosylation activity

The     

Voltammetric reduction of 4-nitroimidazole derivatives: Influence of the N-1 substitution in protic and aprotic media

The voltammetric reduction of 1-methyl- and 1-H- 4-nitroimidazole derivatives was studied in different protic and aprotic media to investigate the influence of the N-1 substitution in the mechanism of reduction, the susceptibility of the nitro group to reduction, and the stability of the nitro radical anion.The elucidation of their voltammetric behavior was carried out using differential pulse polarography and cyclic voltammetry with two different mixed media (Britton-Robinson/ethanol: 70/30 and DMF/citrate: 60/40) and an aprotic media (DMF) at the mercury electrode. In addition, we used UV-vis spectroscopy for the study of their chemistry in solution and quantum-chemical calculations to evaluate LUMO energies, HOMO and LUMO energy gaps, dipole moments and electron affinity, using water and DMF as solvents.The mechanism of reduction was strongly dependent on both the substitution at the N-1 position and the nature of the media. In all media, the methyl-substituted derivative (M-4-NImOH) was always more easily reduced than the demethylated species (H-4-NImOH). On the other hand, the nitro radical anion from M-4-NImOH was more stable than the nitro radical anion from H-4-NImOH.     

Mg–Al hydrotalcite-like compounds containing iron-phthalocyanine complex: effect of aluminum substitution on the complex adsorption features and catalytic activity

Mg–Al hydrotalcite-like materials (Mg_(1−x)Al_x(OH)₂(CO₃)_x/2·nH₂O with x=0.33, 0.25 and 0.20) were used as supports for the immobilization of Fe(III) tetrasulfonated phthalocyanine (FePcTs). Electron paramagnetic resonance (EPR) and X-ray absorption (XANES) spectroscopies were used to characterize the solids after FePcTs adsorption on the hydrotalcite-like materials (HTs). An adsorption study was carried out by monitoring in situ the FePcTs UV/visible electronic spectra during its addition to suspensions of HTs. The results showed that the HT composition controls the nature of adsorbed species: for HTs with higher Al³⁺ contents (x=0.33 and 0.25), the FePcTs was adsorbed mainly in the μ-oxo complex form whereas for HT with x=0.20, non-oxo-bridged FePcTs dimeric species prevailed. The heterogeneous catalytic studies of the HT-FePcTs materials in the oxidation of catechol, using hydrogen peroxide as oxidant, showed an enhanced catalytic activity and longevity, compared to the homogeneous counterpart. The catechol conversion was 63%, 73% and 88% for the materials containing HTs with x=0.20, 0.25 and 0.33, respectively. Therefore, the reactivity of HT-FePcTs materials was improved when the Al³⁺ content in the HTs increases. These catalytic tests associated to the adsorption studies showed that the μ-oxo complex of the FePcTs, mainly adsorbed on the HTs with x=0.25 and 0.33, seems to be the active species in catechol oxidation. These findings suggest that a cooperative effect take place in the HT-FePcTs materials, showing that HTs do not act as an inert support.     

Sulfation: a simple method to enhance the catalytic activity of TS-1 in epoxidation of 1-octene with aqueous hydrogen peroxide

Titanosilicalite (TS-1) has been successfully modified by sulfation to exhibit enhanced catalytic activity in the oxidation of 1-octene with aqueous H₂O₂. A high activity of the sulfated TS-1 was related to modifications of the local environment of Ti active site upon interaction with the .     

Circulating YKL-40 Level,but not CHI3L1 Gene Variants,Is Associated with Atherosclerosis-Related Quantitative Traits and the Risk of Peripheral Artery Disease

YKL-40, a pleotropic cytokine, is emerging as a risk factor and a prognostic predictor of atherosclerotic cardiovascular disease. We attempted to elucidate the genetic, clinical and biochemical correlates of circulating YKL-40 level and, by combining it with CHI3L1 gene variants, with the risk and long-term mortality of peripheral artery disease (PAD). Plasma YKL-40 concentrations were measured in 612 Taiwanese individuals who had no clinically overt systemic disease. Clinical parameters, CHI3L1 gene promoter variants and 18 biomarker levels were analyzed. Eighty-six PAD patients were further enrolled for analysis. Significant associations were found between CHI3L1 genotypes/haplotypes and YKL-40 levels for the health examination subjects (smallest p = 8.36 × 10⁻⁷ for rs4950928 and smallest p = 1.72 × 10⁻¹⁰ for haplotype TGG) and also for PAD patients. For the health examination subjects, circulating YKL-40 level, but not CHI3L1 gene variants, were positively associated with age, smoking, and circulating levels of triglyceride, lipocalin 2 and multiple inflammatory biomarkers and negatively associated with low-density-lipoprotein cholesterol levels. Circulating YKL-40 level is also significantly associated with the risk of PAD (p = 3.3 × 10⁻²³). Circulating YKL40 level, but not CHI3L1 gene promoter variants, is associated with the risk of PAD in Taiwanese. The association of YKL-40 levels with multiple quantitative traits relating to the risk of PAD may provide a molecular basis linking YKL-40 to atherosclerotic cardiovascular disease.     

Studies of the fatty acid specificity of the lipase fromRhizomucor miehei toward 20:1n-9, 20:5n-3, 22:1n-9 and 22:6n-3

The fatty acid specificity of the lipase fromRhizomucor miehei toward 20:1n-9, 20:5n-3, 22:1n-9 and 22:6n-3 has been determined by comparing the alcoholysis (byn-propanol) of various mixtures of C₂₀ and C₂₂ fatty acids (FFA) or the corresponding ethyl esters (FAEE) inn-heptane. For all the fatty acids examined, the degree of conversion was much higher when using FFA rather than FAEE. When comparing the experiments with either single FAEE or FAEE mixtures, it was found for all four fatty acids that the degree of conversion depended on whether the FAEE was alone or together with other fatty acids in the reaction mixture. The lipase showed a strong specificity toward 20:1n-9, whereas the polyunsaturated fatty acids were much poorer substrates, especially 22:6n-3. The degrees of conversion for the two n-3 fatty acids show a clear preference for 20:5n-3 over 22:6n-3, not only when present alone but also in the different mixtures examined. The results obtained in the present experiments therefore suggest that when using the lipase fromR. miehei for enrichment of fish oils with n-3 fatty acids, it should not only be possible to diminish the content of 20:1 and 22:1 present in the outer positions in the triacylglycerols, but also to incorporate relatively more 20:5n-3 than 22:6n-3 into the triglycerides.     

Access of the substrate to the active site of yeast oxidosqualene cyclase: an inhibition and site-directed mutagenesis approach

A structural model of Saccharomyces cerevisiae oxidosqualene cyclase (SceOSC) suggests that some residues of the conserved sequence Pro-Ala-Glu-Val-Phe-Gly (residues 524-529) belong to a channel constriction that gives access to the active-site cavity. Starting from the SceOSC C457D mutant, which lacks the cysteine residue next to the catalytic Asp456 residue Cys457 has been replaced but Asp456 is still there, we prepared two further mutants where the wild-type residues Ala525 and Glu526 were individually replaced by cysteine. These mutants, especially E526C, were very sensitive to the thiol-reacting agent dodecyl-maleimide. Moreover, both the specific activity and the thermal stability of E526C were severely reduced. A similar decrease of the enzyme functionality was obtained by replacing Glu526 with alanine, while substitution with the conservative residues aspartate or glutamine did not alter catalytic activity. Molecular modeling of the yeast wild-type OSC and mutants on the template structure of human OSC confirms that the channel constriction is an important aspect of the protein structure and suggests a critical structural role for Glu526.     

Heterogeneity in recombinant HIV-1 integrase corrected by site-directed mutagenesis: the identification and elimination of a protease cleavage site

Purified recombinant human immunodeficiency virus type 1 (HIV-1) integrase and certain deletion mutants exhibit heterogeneity consistent with proteolysis at a site close to the C-terminus. Electrospray ionization mass spectrometric analysis indicated that proteolytic cleavage generated a protein missing five residues from the C-terminus. PCR mutagenesis of amino acids on either side of the cleavage site identified two changes which were subsequently shown to prevent clipping when proteins were expressed and purified from Escherichia coli: the substitution of Arg284, the residue on the C-terminal side of the cleavage site, by either glycine or lysine. The introduction of either of these mutations into full-length integrase did not affect in vitro 3' processing or strand transfer activities. Thus, the incorporation of either of these mutations is likely to be beneficial when homogeneity of HIV-1 integrase is a concern, as in crystallographic or nuclear magnetic resonance spectroscopic experiments.      

Synthesis and anticancer activity of the (R,S)-benzofused 1,5-oxathiepine moiety tethered to purines through alkylidenoxy linkers

Herein we report the design, synthesis, and anticancer activity of a series of substituted (R,S)-9-[2- or 3-(3,4-dihydro-2H-1,5-benzoxathiepine-3-yloxy)alkyl]-9H-purines. Derivatives with propylenoxy-linked 2',6'-dichloro- and 6'-bromopurines are more active than their respective ethylenoxy-linked purine conjugates. On the other hand, the compound with a propylenoxy-linked 6'-chloropurine is nearly equipotent to the corresponding ethylenoxy-linked conjugate. Our results show that bromo- and chloropurine-conjugated benzoxathiepines containing a propylenoxy linker are able to inhibit PI3 kinase (PI3K) phosphorylation in MCF-7 breast cancer cells, indicating that the activation of eIF2α, together with inhibition of the PI3K pathway, is the mechanism of action by which these compounds effect their antitumor activity in the MCF-7 cell line; apoptosis was induced in a p53-independent manner.     

Characterization and catalytic activity of coal mineral matter: 1. Effect of hydrogen pretreatment and exposure to sulphur and nitrogen compounds

Low temperature ash (LTA) samples prepared from nine US coals were characterized by X-ray diffraction. X-ray fluorescence, and surface area analyses. The results showed that illite, kaolinite, quartz and pyrite are major components in LTAs and that $\text{S}\text{Fe}$ ratios of some LTAs decreased significantly after H₂ treatment implying the occurrence of a partial reduction of pyrite during this treatment. Surface areas of LTAs increased drastically on H₂ treatment but decreased after exposure to sulphur and nitrogen compounds in activity testing. Correlations for the surface areas of LTAs before and after H₂ treatment were found in terms of clay content and element concentrations.     

Crystallization of TS-1 in the presence of alcohols: influence on Ti incorporation and catalytic activity

TS-1 has been synthesized following various recipes of the literature in the presence of alcohols, i.e., from gels that were not evaporated prior to crystallization, which considerably reduces the preparation time of the zeolites. When samples are prepared following a method adapted from the original patent, extra-framework species are observed even for low Ti contents. In contrast, when hydrolysis of the Si and Ti sources is performed in the presence of isopropyl alcohol, all Ti in the zeolite is incorporated in the lattice. However, incorporation is limited to about 1.9 Ti/u.c., even when catalysts are crystallized from gels with relatively high Ti concentrations. These samples are active in the hydroxylation of phenol with H₂O₂ and their activity is comparable with that of conventional catalysts obtained from alcohol-free gels. This revised version was published online in July 2006 with corrections to the Cover Date.