Showing papers on "Substrate (chemistry) published in 1997"
TL;DR: In this article, Al2O3 films with precisely controlled thicknesses and excellent conformality were grown on Si(100) at low temperatures of 350-650 K using sequential surface chemical reactions.
535 citations
TL;DR: Together with a second structurally characterized enzyme state (MCRsilent) containing the heterodisulfide of coenzymes M and B, a reaction mechanism is proposed that uses a radical intermediate and a nickel organic compound.
Abstract: Methyl–coenzyme M reductase (MCR), the enzyme responsible for the microbial formation of methane, is a 300-kilodalton protein organized as a hexamer in an α 2 β 2 γ 2 arrangement. The crystal structure of the enzyme from Methanobacterium thermoautotrophicum , determined at 1.45 angstrom resolution for the inactive enzyme state MCR ox1-silent , reveals that two molecules of the nickel porphinoid coenzyme F 430 are embedded between the subunits α, α′, β, and γ and α′, α, β′, and γ′, forming two identical active sites. Each site is accessible for the substrate methyl–coenzyme M through a narrow channel locked after binding of the second substrate coenzyme B. Together with a second structurally characterized enzyme state (MCR silent ) containing the heterodisulfide of coenzymes M and B, a reaction mechanism is proposed that uses a radical intermediate and a nickel organic compound.
514 citations
TL;DR: A simple, reliable, highly sensitive and quantitative fluorescence microplate-assay of H2O2 from activated leukocytes using a novel horse radish peroxidase (HRP) substrate N-acetyl-3,7-dihydroxyphenoxazine (A6550) is described, which is at least 10-fold higher than with scopoletin as the HRP substrate.
489 citations
TL;DR: A comparison of the substrate-bound and -free forms reveals major conformational differences and provides the first detailed picture of substrate-induced conformational changes in a P450.
Abstract: The substrate-bound structures of two cytochrome P450s, P450cam and P450eryF, are known. While these structures reveal important features that control substrate specificity, the problem of how conformational changes allow for substrate entry and product release remains unsolved. The structure of the haem domain of the bacterial fatty acid hydroxylase, P450BM-3, previously was solved in the substrate-free form. Unlike the substrate-bound P450cam and P450eryF structures, the substrate access channel is open in substrate-free P450BM-3. Here we present the X-ray structure of P450BM-3 at 2.7 A bound with a fatty acid substrate, palmitoleic acid. A comparison of the substrate-bound and -free forms reveals major conformational differences and provides the first detailed picture of substrate-induced conformational changes in a P450.
459 citations
TL;DR: An automated enzyme assay was performed within a microfabricated channel network using resorufin beta-D-galactopyranoside, a substrate that is hydrolyzed to resorUFin, a fluorescent product, and derived Michaelis--Menten constants compared well between an on-chip and a conventional enzyme assay.
Abstract: An automated enzyme assay was performed within a microfabricated channel network. Precise concentrations of substrate, enzyme, and inhibitor were mixed in nanoliter volumes using electrokinetic flow. Reagent dilution and mixing were controlled by regulating the applied potential at the terminus of each channel, using voltages derived from an equivalent circuit model of the microchip. The enzyme β-galactosidase (β-Gal) was assayed using resorufin β-d-galactopyranoside (RBG), a substrate that is hydrolyzed to resorufin, a fluorescent product. Reaction kinetics were obtained by varying the concentration of substrate on-chip and monitoring the production of resorufin using laser-induced fluorescence. Derived Michaelis−Menten constants compared well between an on-chip and a conventional enzyme assay. Bias in the derived Km and kcat was primarily due to the limited solubility of RBG and the associated lack of measurements at substrate concentrations exceeding the Km. A Ki of 8 μM for the inhibitor phenylethyl β...
427 citations
TL;DR: It is proposed that the balance of two opposing effects, one generated by the redox potential difference between a reducing substrate and the type 1 copper of laccase and anothergenerated by the binding of a hydroxide anion to the type 2/type 3 coppers of lAccase, contributes to the pH activity profile of the fungal laccases.
409 citations
TL;DR: In this paper, the state of the art for the heterogeneous enantioselective hydrogenation of α-ketoesters using cinchona modified Pt catalysts and related systems is reviewed.
315 citations
TL;DR: The aim has been to crystallographically demonstrate substrate binding and to locate the two effector-binding sites of RNR R1, and to conclude that the general allosteric effector site, located far from the active site, appears to regulate subunit interactions within the holoenzyme.
242 citations
TL;DR: A steady-state kinetic model describing the dependence of peroxidase activity on hydrogen peroxide concentration is presented and indicates that SBP tends to form more compound III and is catalytically slower than HRP during the oxidation of phenol.
237 citations
TL;DR: The structure of a truncated form of the γ‐subunit of phosphorylase kinase (PHKγt) has been solved in a ternary complex with a non‐hydrolysable ATP analogue and a heptapeptide substrate related to both the natural substrate and to the optimal peptide substrate.
Abstract: The structure of a truncated form of the gamma-subunit of phosphorylase kinase (PHKgammat) has been solved in a ternary complex with a non-hydrolysable ATP analogue (adenylyl imidodiphosphate, AMPPNP) and a heptapeptide substrate related in sequence to both the natural substrate and to the optimal peptide substrate. Kinetic characterization of the phosphotransfer reaction confirms the peptide to be a good substrate, and the structure allows identification of key features responsible for its high affinity. Unexpectedly, the substrate peptide forms a short anti-parallel beta-sheet with the kinase activation segment, the region which in other kinases plays an important role in regulation of enzyme activity. This anchoring of the main chain of the substrate peptide at a fixed distance from the gamma-phosphate of ATP explains the selectivity of PHK for serine/threonine over tyrosine as a substrate. The catalytic core of PHK exists as a dimer in crystals of the ternary complex, and the relevance of this phenomenon to its in vivo recognition of dimeric glycogen phosphorylase b is considered.
187 citations
TL;DR: Gels formed by cross-linking functionalized poly(ethylene glycol) (PEG) and a lysine-containing polypeptide through the action of a natural tissue enzyme, transglutaminase hold potential for forming highly hydrated networks around living cells.
Abstract: We demonstrate formation of a hydrogel network by cross-linking functionalized poly(ethylene glycol) (PEG) and a lysine-containing polypeptide through the action of a natural tissue enzyme, transglutaminase. The enzyme reaction rate using a PEG-modified peptide substrate is the same as the reaction rate for free substrate. Both the ratio and total concentration of the two macromers determine whether gelation will occur and the nature of the gel which forms. Under suitable conditions, clear gels form and swell to give a final composition which is 90% water. Diffusion coefficients of small proteins and albumin in the gel are comparable to those in free solution. Gelation proceeds under mild conditions and thus these gels hold potential for forming highly hydrated networks around living cells.
TL;DR: In this article, an anodic bias of > + 2 V vs NHE was applied to titanium electrodes to generate hydroxyl radicals at the TiO_2 surface, and the branching ratio for these two pathways depends on the nature of the organic substrate.
Abstract: The use of TiO_2 as a photocatalyst for the destruction of organic chemical pollutants in aqueous systems has been extensively studied. One obstacle to the effective utilization of these systems is the relatively inefficient use of the solar spectrum by the photocatalyst. In addition, light delivery to the photocatalyst can be impeded by UV-absorbing components in mixed effluent streams. We present a novel use of TiO_2 as a catalyst for the oxidative degradation of organic compounds in water that uses a potential source instead of light to generate reactive oxidants. Application of an anodic bias of >+2 V vs NHE to titanium electrodes coated with niobium-doped, polycrystalline TiO_2 particles electrochemically generates hydroxyl radicals at the TiO_2 surface. This process has been demonstrated to efficiently degrade a variety of environmentally important pollutants. In addition, these electrodes offer a unique opportunity to probe mechanistic questions in TiO_2 catalysis. By comparing substrate degradation rates with increases in current density upon substrate addition, the extent of degradation due to direct oxidation and •OH oxidation can be quantified. The branching ratio for these two pathways depends on the nature of the organic substrate. Formate is shown to degrade primarily via a hydroxyl radical mechanism at these electrodes, whereas the current increase data for compounds such as 4-chlorocatechol indicate that a higher percentage of their degradation may occur through direct oxidation. In addition, the direct oxidation pathway is shown to be more important for 4-chlorocatechol, a strongly adsorbing substrate, than for 4-chlorophenol, which does not adsorb strongly to TiO_2.
TL;DR: The overexpressed BTL2 lipase shows a strong tendency to aggregate and under certain conditions, a direct relationship was found between the molecular mass of the lipase aggregates and the increase in activity upon the addition of 1% (w/v) sodium cholate.
TL;DR: The three-dimensional structure of the substrate analogue binary complex could give important insight into the molecular mechanism of this enzyme, which defies a simple interpretation of their roles in resistance.
TL;DR: Fluorescence microscopy with a cooled CCD imaging system is used to visualize the spatial distribution of enzyme immobilization sites (indicated by fluorescence from Texas Red-labeled avidin) across the carbon surface and the viability of the enzyme attached to the surface was demonstrated by imaging the distribution of an insoluble, fluorescent product.
Abstract: Micrometer-sized domains of a carbon surface are modified to allow derivatization to attach redox enzymes with biotin/avidin technology. These sites are spatially segregated from and directly adjacent to electron transfer sites on the same electrode surface. The distance between these electron transfer sites and enzyme-loaded domains must be kept to a minimum (e.g., less than 5 μm) to maintain the fast response time and high sensitivity required for the measurement of neurotransmitter dynamics. This is accomplished through the use of photolithographic attachment of photobiotin using an interference pattern from a UV laser generated at the electrode surface. This will allow the construction of microscopic arrays of active enzyme sites on a carbon fiber substrate while leaving other sites underivatized to facilitate electron transfer reactions of redox mediators, thus maximizing enzyme activity and detection of the enzyme mediator. The ultimate sensitivity of these sensors will be realized only through care...
TL;DR: The structure of VAO complexed with the inhibitor 4-(1-heptenyl)phenol shows that the catalytic cavity is completely filled by the inhibitor, explaining why alkylphenols bearing aliphatic substituents longer than seven carbon atoms do not bind to the enzyme.
TL;DR: Evidence is provided that the zinc ion plays an important catalytic role in FTase, most likely by activation of the cysteine thiol of the protein substrate for nucleophilic attack on the isoprenoid.
TL;DR: The data suggest that hydrolysis of MgATP is required to activate the enzyme; in the absence of substrate the energy involved in this activation dissipates, and the excited state isrequired to overcome the high activation energy associated with the loss of the aromatic character and/or to render ring reduction irreversible.
Abstract: An enzyme was recently described, benzoyl-CoA reductase (dearomatizing), which catalyses the ATP-driven reduction of the aromatic ring of benzoyl-CoA yielding a non-aromatic CoA thioester, ADP and phosphate [Boll, M. & Fuchs, G. (1995) Eur. J. Biochem. 234, 921-933]. The 170-kDa enzyme consists of four different subunits and contains approximately 12 Fe and acid-labile sulfur/mol. Benzoyl-CoA reductase exhibits ATPase activity in the absence of substrate. It is shown that only the reduced form of this iron-sulfur protein has ATPase activity. ATPase activity is reversibly lost when the enzyme is oxidized by thionine; reduction of the enzyme fully restores ATPase and ring-reduction activity. 2 mol ATP are hydrolyzed/2 mol electrons transferred in the course of the reaction. The product ADP acts as competitive inhibitor (Ki = 1.1 mM) for ATP in benzoyl-CoA reduction; ADP inhibits ATPase activity to the same extent as ring-reduction activity. EPR investigation of the dithionite-reduced enzyme suggested the presence of two separate [2Fe-2S] clusters and two interacting [4Fe-4S] clusters. Addition of MgATP to the reduced enzyme resulted in a new isotropic signal at g = 5.15 and a weak signal at g = 12; in controls with MgADP only a minor signal at g = 5.15 was observed. The positions, shapes and temperature dependencies of these MgATP-induced signals are indicative for excited states of a S = 7/2 spin multiplet. The [2Fe-2S] signals were not affected by ATP, but one of the [4Fe-4S] clusters became slowly oxidized. Addition of both benzoyl-CoA and MgATP resulted in a major oxidation of the iron-sulfur clusters accompanied by the appearance of some minor signals of unknown origin in the g = 2.037-1.96 region. Neither the benzoyl-CoA plus MgATP-oxidized nor the thionine-oxidized enzyme showed the ATP-dependent formation of the high-spin signals of the reduced enzyme. At present we hypothesize that the S = 7/2 signal is due to an ATP-induced change of one of the [4Fe-4S] clusters. The data suggest that hydrolysis of MgATP is required to activate the enzyme; in the absence of substrate the energy involved in this activation dissipates. MgATP-driven formation of this excited state of the reduced enzyme rather than transfer of electrons from the reduced enzyme to the aromatic substrate appears to be the rate-limiting step in the catalytic cycle. We suggest that the excited state is required to overcome the high activation energy associated with the loss of the aromatic character and/or to render ring reduction irreversible.
TL;DR: The results showed that the hydrophilic property and the existing mesopores of DAY played important roles in the enzyme immobilization and the resulting biosensor exhibited good reproducibility and selectivity.
Abstract: A new approach to construct an amperometric biosensor is described. Without using bovine serum albumin−glutaraldehyde, glucose oxidase (GOx) was immobilized on a dealuminized Y zeolite (DAY)-modified platinum electrode to construct a glucose sensor. The large specific surface area of the zeolite substrate resulted in high enzyme loading. The immobilized GOx in this manner was stable and could maintain its high activity for at least 3 months. The interactions between the zeolite and the enzyme were investigated by means of Fourier transform infrared spectra, and the pore distribution and the surface acid property of DAY were preliminarily studied. The results showed that the hydrophilic property and the existing mesopores of DAY played important roles in the enzyme immobilization. This resulting biosensor exhibited good reproducibility and selectivity, owing to the uniform pore structure and unique ion-exchange property of the zeolite. The biosensor responded rapidly to glucose in the linear range from 2.0...
TL;DR: The three-dimensional structure of the quaternary complex of Saccharomyces cerevisiae transketolase, thiamin diphosphate, Ca2+, and the acceptor substrate erythrose-4-phosphate has been determined, suggesting a role for this amino acid in substrate binding and catalysis.
TL;DR: The present structure underlines the central role of the carbamylated lysine 201 in both activation and catalysis, and completes available structural information for the proposal on the mechanism of the enzyme.
TL;DR: In this paper, the advantages of the coupling of sol-gel enzyme electrode preparation processes with the dispersion of electrocatalytic metal centers are described. And the performance of solgel-derived metal-doped enzyme strips are discussed.
Abstract: The advantages which accrue from the coupling of sol-gel enzyme electrode preparation processes with the dispersion of electrocatalytic metal centers are described. In particular, ruthenium-dispersed sol-gel carbon thick-film bioelectrodes couple the low-temperature curing process with the elimination of contributions from easily oxidizable interfering species. The remarkable selectivity towards the glucose substrate is combined with a greatly enhanced sensitivity. Factors influencing the performance of these sol-gel-derived metal-doped enzyme strips are discussed.
TL;DR: The effect of water activity on V(max) showed that the enzyme activity in general increased with increasing amounts of water for the three enzymes, shown both for esterification and hydrolysis reactions catalyzed by R. arrhizus lipase.
Abstract: Catalytic activity of lipases (from Rhizopus arrhizus, Canadida rugosa, and Pseudomonas sp. was studied in organic media, mainly diisopropyl ether. The effect of water activity (a(w)) on V(max) showed that the enzyme activity in general increased with increasing amounts of water for the three enzymes. This was shown both for esterification and hydrolysis reactions catalyzed by R. arrhizus lipase. In the esterification reaction the K(m) for the acid substrate showed a slight increase with increasing water activities. On the other hand, the K(m) for the alcohol substrate increased 10-20-fold with increasing water activity. The relative changes in K(m) were shown to be independent of the enzyme studied and solvent used. The effect was attributed to the increasing competition of water as a nucleophile for the acyl-enzyme at higher water activities. In a hydrolysis reaction the K(m) for the ester was also shown to increase as the water activity increased. The effect of water in this case was due to the fact that increased concentration of one substrate (water), and thereby increased saturation of the enzyme, will increase the apparent K(m) of the substrate (ester) to be determined. This explained why the hydrolysis rate decreased with increasing water activity at a fixed, low ester concentration. The apparent V(max) for R. arrhizus lipase was similar in four of six different solvents that were tested; exceptions were toulene and trichloroethylene, which showed lower values. The apparent K(m) for the alcohol in the solvents correlated with the hydrophobicity of the solvent, hydrophobic solvents giving lower apparent K(m). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 798-806, 1997.
TL;DR: Sensors constructed by the adsorption and dip coating of L-glutamate oxidase had a fast response time, high sensitivity to Glu, and possessed selectivity characteristics suggesting that monitoring Glu in biological tissues in vivo may be feasible.
Abstract: An amperometric biosensor for L-glutamic acid (Glu) was constructed by the adsorption and dip coating of L-glutamate oxidase (GluOx, 200 U ml–1 phosphate buffer, pH 7.4) onto 60-µm radius Teflon-coated Pt wire (1 mm exposed length). The enzyme was then trapped on the surface by electropolymerisation of o-phenylenediamine that also served to block electroactive interference. This procedure afforded electrodes with similar substrate sensitivity compared with the classical approach of immobilising enzyme from a solution of monomer, and represents an approximately 10000-fold increase in the yield of biosensors from a batch of enzyme. A number of strategies were examined to enhance the sensitivity and selectivity of the Pt/PPD/GluOx sensors operating at 0.7 V versus SCE. Pre-coating the Pt with lipid and incorporation of the protein bovine serum albumin into the polymer matrix were found to improve the performance of the electrode. The sensors had a fast response time, high sensitivity to Glu, with an LOD of about 0.3 µmol l–1, and possessed selectivity characteristics suggesting that monitoring Glu in biological tissues in vivo may be feasible.
TL;DR: As a first step in the development of a reporter system for gene expression in halophilic archaea, a beta-galactosidase was purified 140-fold from Haloferax alicantei (previously phenon K, strain Aa2.2).
TL;DR: The catalytic activity was found to be highly dependent on the orientation of the complex within the film and the observed rate dependence on temperature strongly implicate the molecular order of a metal complex as an important dimension in catalysis.
Abstract: Catalytically active Langmuir-Blodgett films of a rhodium complex were prepared and characterized to determine the possible effect of the molecular order of metal complexes on catalytic activity. The hydrogenation of carbon-oxygen double bonds was used as a model reaction. The complex in solution exhibited low catalytic activity, whereas it was highly active in the film. The catalytic activity was found to be highly dependent on the orientation of the complex within the film. The reactions were also highly selective with regard to the substrate. These observations and the observed rate dependence on temperature strongly implicate the molecular order of a metal complex as an important dimension in catalysis.
TL;DR: How P-glycoprotein recognizes a wide variety of compounds and how it carries its substrates across membranes is reviewed and some amino acid residues in the putative transmembrane domain (TM) 1 together with TM5-6 and TM11-12 may help to govern substrate specificity.
TL;DR: The NAD(P)H-dependent enzymatic activity within living Zygosaccharomyces rouxii was utilized to accomplish this reaction and the reaction was scaled-up to a volume of 300 l by utilizing a commercially available agitated filter as a reactor.
TL;DR: In this article, the TiO_2-catalyzed photodegradation of a strongly adsorbing substrate, 4-chlorocatechol, has been investigated as a function of solution concentration and pH at illuminated polycrystalline TiO-2 electrodes operated at a constant current density.
Abstract: The TiO_2-catalyzed photodegradation of a strongly adsorbing substrate, 4-chlorocatechol, has been investigated as a function of solution concentration and pH at illuminated polycrystalline TiO_2 electrodes operated at a constant current density. The results are compared to the previously determined sorption behavior of 4-chlorocatechol. The initial rates of 4-chlorocatechol photodegradation measured at pH 3, 5, and 8 and solution concentrations of 20, 50, and 200 μM show a linear correlation with the concentration of the sorbed substrate.
TL;DR: The results indicate that the substrate reactivity falls only modestly as conversion increases, but the restart rate is still higher than the rate of the uninterrupted hydrolysis, particularly at high conversion.
Abstract: In an effort to better understand the role of the substrate in the rapid fall off in the rate of enzymatic hydrolysis of cellulose with conversion, substrate reactivity was measured as a function of conversion. These measurements were made by interrupting the hydrolysis of pretreated wood at various degrees of conversion; and, after boiling and washing, restarting the hydrolysis in fresh buffer with fresh enzyme. The comparison of the restart rate per enzyme adsorbed with the initial rate per enzyme adsorbed, both extrapolated back to zero conversion, provides a measurement of the substrate reactivity without the complications of product inhibition or cellulase inactivation. The results indicate that the substrate reactivity falls only modestly as conversion increases. However, the restart rate is still higher than the rate of the uninterrupted hydrolysis, particularly at high conversion. Hence we conclude that the loss of substrate reactivity is not the principal cause for the long residence time required for complete conversion. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng56: 650–655, 1997.