Showing papers on "Substrate (chemistry) published in 1999"
TL;DR: In this review the diversity of lipoxygenase expression will be highlighted and the several facets of lip oxygengenase function considered, concluding with a discussion of issues related to the acquisition of substrate in a cellular environment.
1,268 citations
TL;DR: Immobilization methods range from binding to prefabricated carrier materials to packaging in enzyme crystals or powders, which reduce the reaction rates and product yields and must be minimized in order to increase their competitiveness for technical applications.
393 citations
TL;DR: Two X-ray structures of CGTase complexes are determined, which give evidence for substrate distortion and the covalent character of the intermediate and for the first time show, in atomic detail, how catalysis in the α-amylase family proceeds by the concerted action of all active site residues.
Abstract: Cyclodextrin glycosyltransferase (CGTase) is an enzyme of the α-amylase family, which uses a double displacement mechanism to process α-linked glucose polymers. We have determined two X-ray structures of CGTase complexes, one with an intact substrate at 2.1 A resolution, and the other with a covalently bound reaction intermediate at 1.8 A resolution. These structures give evidence for substrate distortion and the covalent character of the intermediate and for the first time show, in atomic detail, how catalysis in the α-amylase family proceeds by the concerted action of all active site residues.
375 citations
TL;DR: In this paper, the effect of free fatty acid (FFA) content on the susceptibility to thermooxidative degeneration of vegetable oils was determined by Rancimat analysis.
Abstract: The effect of free fatty acid (FFA) content on the susceptibility to thermooxidative degeneration of vegetable oils was determined by Rancimat analysis. A prooxidant effect of FFA was observed in all filtered oils, independently of lipidic substrate and of its state of hydrolytic and oxidative alteration. The intensity of this effect was related to FFA concentration, but regression analysis of the experimental data did not show a general correlation law between FFA concentration and induction time (I
t). Different results were obtained for freshly processed virgin olive oils, characterized by postpressing natural suspension-dispersion: opposite behavior was observed of FFA content as regards oxidative stability, depending on the presence of suspended-dispersed material. This fact is of interest because the dispersed particles play a double stabilizing effect on both oxidative and hydrolytic degradation. These results showed that avoidance of oil filtration is highly desirable to extend olive oil’s shelf life.
315 citations
TL;DR: Water-repellent surfaces have been prepared by exposing Si substrates with a hydroxylated surface oxide to fluoroalkyl silane (FAS) vapor as mentioned in this paper.
Abstract: Water-repellent surfaces have been prepared by exposing Si substrates with a hydroxylated surface oxide to fluoroalkyl silane (FAS) vapor. Since this chemical vapor surface modification (CVSM) is based on the chemical reaction between organosilane molecules and hydroxyl groups at the oxide surface, prior to CVSM, the substrate surface was completely hydroxylated by irradiating in air with a 172-nm ultraviolet light until the water contact angle of the surface became almost 0°. Under atmospheric pressure, the substrate was then exposed to vapor of an FAS precursor, that is, one of three types of FAS having different perfluoroalkyl chain lengths [CF3(CF2)nCH2CH2Si(OCH3)3, where n = 0, 5, or 7, referred to as FAS-3, FAS-13, and FAS-17, respectively]. The FAS molecules chemically reacted with the hydroxyl groups on the substrate surface and adsorbed onto it, forming a thin layer of less than 2 nm in thickness. The water repellency of the substrate surface increased with an increase in perfluoroalkyl chain len...
299 citations
TL;DR: Simulation of the complex between the reduced enzyme and the product, delta-gluconolactone, has provided an explanation for the lack of product inhibition by the lactone.
Abstract: Glucose oxidase is a flavin-dependent enzyme which catalyses the oxidation of β-d-glucose by molecular oxygen to δ-gluconolactone and hydrogen peroxide. The structure of the enzyme from Aspergillus niger, previously refined at 2.3 A resolution, has been refined at 1.9 A resolution to an R value of 19.0%, and the structure of the enzyme from Penicillium amagasakiense, which has 65% sequence identity, has been determined by molecular replacement and refined at 1.8 A resolution to an R value of 16.4%. The structures of the partially deglycosylated enzymes have an r.m.s. deviation of 0.7 A for main-chain atoms and show four N-glycosylation sites, with an extended carbohydrate moiety at Asn89. Substrate complexes of the enzyme from A. niger were modelled by force-field methods. The resulting model is consistent with results from site-directed mutagenesis experiments and shows the β-d-glucose molecule in the active site of glucose oxidase, stabilized by 12 hydrogen bonds and by hydrophobic contacts to three neighbouring aromatic residues and to flavin adenine dinucleotide. Other hexoses, such as α-d-glucose, mannose and galactose, which are poor substrates for the enzyme, and 2-deoxy-d-glucose, form either fewer bonds or unfavourable contacts with neighbouring amino acids. Simulation of the complex between the reduced enzyme and the product, δ-gluconolactone, has provided an explanation for the lack of product inhibition by the lactone.
288 citations
TL;DR: The crystal structure of InhA in complex with NAD+ and a C16 fatty acyl substrate,trans-2-hexadecenoyl-(N-acetylcysteamine)-thioester, reveals that the substrate binds in a general “U-shaped” conformation, consistent with the ability of InHA to recognize longer chain fatty acy substrates.
263 citations
01 Jan 1999
TL;DR: The structure of the enzyme from Aspergillus niger, previously re®ned at 2.3 AE resolution, has been re´ned at 1.9 AE resolution to an R value of 19.0% as discussed by the authors.
Abstract: # 1999 International Union of Crystallography Printed in Denmark ± all rights reserved Glucose oxidase is a avin-dependent enzyme which catalyses the oxidation of -d-glucose by molecular oxygen to -gluconolactone and hydrogen peroxide. The structure of the enzyme from Aspergillus niger, previously re®ned at 2.3 AE resolution, has been re®ned at 1.9 AE resolution to an R value of 19.0%, and the structure of the enzyme from Penicillium amagasakiense, which has 65% sequence identity, has been determined by molecular replacement and re®ned at 1.8 AE resolution to an R value of 16.4%. The structures of the partially deglycosylated enzymes have an r.m.s. deviation of 0.7 AE for main-chain atoms and show four N-glycosylation sites, with an extended carbohydrate moiety at Asn89. Substrate complexes of the enzyme from A. niger were modelled by force-®eld methods. The resulting model is consistent with results from site-directed mutagenesis experiments and shows the -d-glucose molecule in the active site of glucose oxidase, stabilized by 12 hydrogen bonds and by hydrophobic contacts to three neighbouring aromatic residues and to avin adenine dinucleotide. Other hexoses, such as -d-glucose, mannose and galactose, which are poor substrates for the enzyme, and 2-deoxy-d-glucose, form either fewer bonds or unfavourable contacts with neighbouring amino acids. Simulation of the complex between the reduced enzyme and the product, -gluconolactone, has provided an explanation for the lack of product inhibition by the lactone. Received 25 November 1998 Accepted 2 March 1999
236 citations
TL;DR: In this article, the authors investigated the efficiency of using soybean peroxidase (SBP) to remove several different phenolic compounds from unbuffered synthetic wastewater, including parent phenol, chlorinated phenols, cresols, 2,4-dichlorophenol and 4,4′-isopropylidenediphenol (commonly known as bisphenol A).
228 citations
TL;DR: This is the first crystallographic indication of the 'base-on' mode of cobalamin binding, which seems to favor homolytic cleavage of the cobalt-carbon bond and therefore to favor radical enzyme catalysis.
210 citations
TL;DR: 6-FAM-dArUdAdA-6-TAMRA is the most sensitive known substrate for detecting ribo-nucleolytic activity and enables a simple protocol for the rapid determination of the inhibition constant for competitive inhibitors such as uridine 3'-phosphate and adenosine 5'-diphos-phate.
Abstract: A substrate for a hypersensitive assay of ribonucleolytic activity was developed in a systematic manner This substrate is based on the fluorescence quenching of fluorescein held in proximity to rhodamine by a single ribonucleotide embedded within a series of deoxynucleotides When the substrate is cleaved, the fluorescence of fluorescein is manifested The optimal substrate is a tetranucleotide with a 5',6-carboxyfluorescein label (6-FAM) and a 3',6-carboxy-tetramethylrhodamine (6-TAMRA) label: 6-FAM-dArUdAdA-6-TAMRA The fluorescence of this substrate increases 180-fold upon cleavage Bovine pancreatic ribonuclease A (RNase A) cleaves this substrate with a k (cat)/ K (m)of 36 x 10(7)M(-1)s(-1) Human angiogenin, which is a homolog of RNase A that promotes neovascularization, cleaves this substrate with a k (cat)/ K (m)of 3 3 x 10(2)M(-1)s(-1) This value is >10-fold larger than that for other known substrates of angio-genin With these attributes, 6-FAM-dArUdAdA-6-TAMRA is the most sensitive known substrate for detecting ribo-nucleolytic activity This high sensitivity enables a simple protocol for the rapid determination of the inhibition constant ( K (i)) for competitive inhibitors such as uridine 3'-phosphate and adenosine 5'-diphos-phate
TL;DR: The relative distribution of the modes of hydrocarbon uptake, used by bacteria of the environment for the degradation of long chain alkanes, has been evaluated and suggested the existence of two distinct alkane transfer mechanisms in this group.
Abstract: The relative distribution of the modes of hydrocarbon uptake, used by bacteria of the environment for the degradation of long-chain alkanes, has been evaluated. The first mode of uptake, direct interfacial accession, involves contact of cells with hydrocarbon droplets. In the second mode, biosurfactant-mediated transfer, cell contact takes place with hydrocarbons emulsified or solubilized by biosurfactants. Sixty-one strains growing on hexadecane were isolated from polluted and non-polluted soils and identified. The majority (61%) belonged to the Corynebacterium-Mycobacterium-Nocardia group. Criteria selected for characterizing hexadecane uptake were cell hydrophobicity, interfacial and surface tensions and production of glycolipidic extracellular biosurfactants. These properties were determined in flask cultures on an insoluble (hexadecane) and on a soluble (glycerol or succinate) carbon source for a subset of 23 representative strains. Exclusive direct interfacial uptake was utilized by 47% of studied strains. A large proportion of strains (53%) produced biosurfactants. The data on cellular hydrophobicity suggested the existence of two distinct alkane transfer mechanisms in this group. Accordingly, tentative assignments of biosurfactant-mediated micellar transfer were made for 11% of the isolated strains, and of biosurfactant-enhanced interfacial uptake for 42%.
TL;DR: The role of residue Tyr212 as a key catalytic residue, suggested by the crystal structure of the inhibitor complex, is confirmed by mutagenesis results and a structural model of catalysis is presented based on these results.
TL;DR: In this paper, a new colloidal processing method for near net shaping ceramic components is outlined, which relies on electrostatic stabilized ceramic suspensions and it’s destabilization by time delayed in situ reactions.
TL;DR: The structure of human glyoxalase II was solved initially by single isomorphous replacement with anomalous scattering and refined at a resolution of 1.9 A, and the reaction mechanism may also have implications for the action of metallo-beta-lactamases.
TL;DR: Histomorphometric analysis conducted on samples inserted in the cancellous bone of distal femoral epiphysis of Sprague-Dawley rats gave the following results: Affinity index (AI%) data proving the surface osteconductive properties of non-anodized acid etched Ti indicated that hydroxyapatite allowed a higher bone to implant contact respect to Ti only.
TL;DR: The structure represents a novel example of how an enzyme acquires a different substrate profile and a catalytic versatility from a common active site and represents a framework for explaining the catalytic activities of transglycosylation and hydrolysis of α-d-(1,6)-glycosidic bond.
TL;DR: The catalytic mechanism of metallo-beta-lactamase from Bacteroides fragilis, a dinuclear Zn(II)-containing enzyme responsible for multiple antibiotic resistance, has been investigated by using nitrocefin as a substrate, showing a novel anionic species bound to the enzyme through a Zn-acyl linkage and contains a negatively charged nitrogen leaving group.
Abstract: The catalytic mechanism of metallo-beta-lactamase from Bacteroides fragilis, a dinuclear Zn(II)-containing enzyme responsible for multiple antibiotic resistance, has been investigated by using nitrocefin as a substrate. Rapid-scanning and single-wavelength stopped-flow studies revealed the accumulation during turnover of an enzyme-bound intermediate with intense absorbance at 665 nm (epsilon = 30 000 M(-1) cm(-1)). The proposed minimum kinetic mechanism for the B. fragilis metallo-beta-lactamase-catalyzed nitrocefin hydrolysis [Wang, Z., and Benkovic, S. J. (1998) J. Biol. Chem. 273, 22402-22408] was confirmed, and more accurate kinetic parameters were obtained from computer simulations and fitting. The intermediate was shown to be a novel anionic species bound to the enzyme through a Zn-acyl linkage and contains a negatively charged nitrogen leaving group. This is the first time such an intermediate was observed in the catalytic cycle of a Zn(II)-containing hydrolase and is evidence for a unique beta-lactam hydrolysis mechanism in which the amine can leave as an anion; prior protonation is not required. The electrostatic interaction between the negatively charged intermediate and the positively charged dinuclear Zn(II) center of the enzyme is important for stabilization of the intermediate. The catalytic reaction was accelerated in the presence of exogenous nucleophiles or anions, and neither the product nor the enzyme was modified during turnover, indicating that a Zn-bound hydroxide (rather than Asp-103) is the active site nucleophile. On the basis of all the information on hand, a catalytic mechanism of the B. fragilis metallo-beta-lactamase is proposed.
TL;DR: The present results demonstrate clearly the importance of the cumulative effects of residues remote from the active site and represent a new line of approach to the redesign of enzyme activity.
TL;DR: The crystallographic results show that one of the tunnel-forming loops in Cel6A is sensitive to modifications at the active site, and is able to take on a number of different conformations.
TL;DR: X-ray crystal structures of three species related to the oxidative half of the reaction of the copper-containing quinoprotein amine oxidase from Escherichia coli have been determined, revealing the site of bound dioxygen and the proton transfer pathways involved in oxygen reduction.
Abstract: X-ray crystal structures of three species related to the oxidative half of the reaction of the copper-containing quinoprotein amine oxidase from Escherichia coli have been determined. Crystals were freeze-trapped either anaerobically or aerobically after exposure to substrate, and structures were determined to resolutions between 2.1 and 2.4 angstroms. The oxidation state of the quinone cofactor was investigated by single-crystal spectrophotometry. The structures reveal the site of bound dioxygen and the proton transfer pathways involved in oxygen reduction. The quinone cofactor is regenerated from the iminoquinone intermediate by hydrolysis involving Asp383, the catalytic base in the reductive half-reaction. Product aldehyde inhibits the hydrolysis, making release of product the rate-determining step of the reaction in the crystal.
TL;DR: It is hypothesized that H608V exchange may alter the orientation of the substrate at the active site and/or its steric configuration in such a way that a stereospecific dioxygen insertion at C-9 may exclusively take place.
Abstract: Multiple lipoxygenase sequence alignments and structural modeling of the enzyme/substrate interaction of the cucumber lipid body lipoxygenase suggested histidine 608 as the primary determinant of positional specificity. Replacement of this amino acid by a less-space-filling valine altered the positional specificity of this linoleate 13-lipoxygenase in favor of 9-lipoxygenation. These alterations may be explained by the fact that H608V mutation may demask the positively charged guanidino group of R758, which, in turn, may force an inverse head-to-tail orientation of the fatty acid substrate. The R758L+H608V double mutant exhibited a strongly reduced reaction rate and a random positional specificity. Trilinolein, which lacks free carboxylic groups, was oxygenated to the corresponding (13S)-hydro(pero)xy derivatives by both the wild-type enzyme and the linoleate 9-lipoxygenating H608V mutant. These data indicate the complete conversion of a linoleate 13-lipoxygenase to a 9-lipoxygenating species by a single point mutation. It is hypothesized that H608V exchange may alter the orientation of the substrate at the active site and/or its steric configuration in such a way that a stereospecific dioxygen insertion at C-9 may exclusively take place.
TL;DR: An extracellular lipase of Streptomyces rimosus R6-554W was isolated from the culture filtrate by column chromatography and was shown to be a monomeric, basic protein active toward triolein and p-nitrophenyl esters, with preference for those with medium size acyl chain length.
TL;DR: In this paper, the structure of arginase from Bacillus caldovelox at pH 5.6 and pH 8.5 was investigated and the results indicated a common mode of ligand binding, which places the substrate adjacent to the dimanganese centre.
TL;DR: An inverse correlation was shown between AAH stereoselectivity and the brain cannabinoid receptor affinity as the enantiomers with high receptor affinity displayed low susceptibility to hydrolysis by AAH.
Abstract: Anandamide amidohydrolase (AAH) catalyzes the hydrolysis of arachidonylethanolamide (anandamide), an endogenous cannabinoid receptor ligand. To delineate the structural requirements of AAH substrates, rat brain microsomal AAH hydrolysis of a series of anandamide congeners was studied using two reverse-phase high-performance liquid chromatography (RP-HPLC) assays developed in our laboratory. Arachidonamide (1) was found to be the best substrate with an apparent Km of 2.34 mM and a Vmax of 2.89 nmol/min/mg of protein. Although anandamide (2) has a similar Km value, its Vmax is approximately one-half that of arachidonamide. N,N-Bis(2-hydroxyethyl)arachidonamide (3) was not hydrolyzed, suggesting specificity for unsubstituted or mono-N-substituted arachidonamides. Analogues with a methyl group at the 1‘-position of the ethanolamido headgroup were also found to have greater resistance to enzymatic turnover and therefore increased metabolic stability. The enzyme exhibited high stereoselectivity as the rate of h...
TL;DR: Evidence was presented that substrate induction was a major regulatory mechanism and the keratinase biosynthesis was not completely repressed by addition of other carbon (glucose) and nitrogen (NH4C1) sources.
Abstract: The keratinase production by the thermophilic actinomycete strain Thermoactinomyces candidus was induced by sheep wool as the sole source of carbon and nitrogen in the cultivation medium. For complete digestion of wool by the above strain, both keratinolytic serine proteinase and cellular reduction of disulfide bonds were involved. Evidence was presented that substrate induction was a major regulatory mechanism and the keratinase biosynthesis was not completely repressed by addition of other carbon (glucose) and nitrogen (NH4Cl) sources. The enzyme was purified 62-fold by diethylaminoethyl - anion exchange and Sephadex G-75 gel permeation chromatographies. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis indicated that the purified keratinase is a monomeric enzyme with a molecular mass of 30 kDa. The pH and temperature optima were determined to be 8.6 and 70°C, respectively. The purified thermophilic keratinase catalyses the hydrolysis of a broad range of substrates and displays higher proteoly...
Patent•
08 Oct 1999
TL;DR: In this article, a method for manufacturing a thin film includes the steps of loading a substrate into a reaction chamber, and terminating the surface of the substrate loaded into the reaction chamber by a specific atom.
Abstract: A method for manufacturing a thin film includes the steps of loading a substrate into a reaction chamber, and terminating the surface of the substrate loaded into the reaction chamber by a specific atom. A first reactant is chemically adsorbed on the terminated substrate by injecting the first reactant into the reaction chamber including the terminated substrate. After removing the first reactant physically adsorbed into the terminated substrate, a solid thin film is formed through chemical exchange or reaction of the chemically adsorbed first reactant and a second reactant by injecting the second reactant into the reaction chamber. According to the thin film manufacturing method according to the present invention, it is possible to grow a thin film on the substrate in a state in which the no or little impurities and physical defects are generated in the thin film and interface of the thin film.
TL;DR: The position of Tyralpha89 close to the substrate explains the stereochemical selectivity of the enzyme for (2R)-methylmalonyl-CoA.
Abstract: X-ray crystal structures of methylmalonyl-CoA mutase in complexes with substrate methylmalonyl-CoA and inhibitors 2-carboxypropyl-CoA and 3-carboxypropyl-CoA (substrate and product analogues) show that the enzyme-substrate interactions change little during the course of the rearrangement reaction, in contrast to the large conformational change on substrate binding. The substrate complex shows a 5'-deoxyadenine molecule in the active site, bound weakly and not attached to the cobalt atom of coenzyme B12, rotated and shifted from its position in the substrate-free adenosylcobalamin complex. The position of Tyralpha89 close to the substrate explains the stereochemical selectivity of the enzyme for (2R)-methylmalonyl-CoA.
TL;DR: The results imply an extended bound conformation, highlight a role for the presence, position, and stereochemistry of a delta cis double bond, and suggest little apparent role for C11-C18/C22 of the fatty acid amide substrates.