Showing papers on "Substrate (chemistry) published in 1992"

Electrical Connection of Enzyme Redox Centers to Electrodes

Abstract: : Electrically insulating proteins can be made redox-conducting through incorporation of a high density of electron relaying redox centers. Electrons diffuse in the resulting redox conductors by self-exchange between identical and electron transfer between different relaying centers. When the self-exchange rate of the relays and their density are high, the flux of electrons through a 1 micrometer thick film of a 3-dimensional macromolecular network can match or exceed the rate of supply of electrons to or from the ensemble of enzyme molecules covalently bound to it. The network now molecularly wires the enzyme molecules to the electrode and the current measures the turnover of the wired enzyme molecules. When the enzyme turnover is substrate-flux, i.e. concentration limited, the current increases with the concentration of the substrate.

Converting Trypsin to Chymotrypsin: The Role of Surface Loops

Abstract: Trypsin (Tr) and chymotrypsin (Ch) have similar tertiary structures, yet Tr cleaves peptides at arginine and lysine residues and Ch prefers large hydrophobic residues. Although replacement of the S1 binding site of Tr with the analogous residues of Ch is sufficient to transfer Ch specificity for ester hydrolysis, specificity for amide hydrolysis is not transferred. Trypsin is converted to a Ch-like protease when the binding pocket alterations are further modified by exchange of the Ch surface loops 185 through 188 and 221 through 225 for the analogous Tr loops. These loops are not structural components of either the S1 binding site or the extended substrate binding sites. This mutant enzyme is equivalent to Ch in its catalytic rate, but its substrate binding is impaired. Like Ch, this mutant utilizes extended substrate binding to accelerate catalysis, and substrate discrimination occurs during the acylation step rather than in substrate binding.

Total chemical synthesis of a D-enzyme: the enantiomers of HIV-1 protease show reciprocal chiral substrate specificity [corrected]

Abstract: The D and L forms of the enzyme HIV-1 protease have been prepared by total chemical synthesis. The two proteins had identical covalent structures. However, the folded protein-enzyme enantiomers showed reciprocal chiral specificity on peptide substrates. That is, each enzyme enantiomer cut only the corresponding substrate enantiomer. Reciprocal chiral specificity was also evident in the effect of enantiomeric inhibitors. These data imply that the folded forms of the chemically synthesized D- and L-enzyme molecules are mirror images of one another in all elements of the three-dimensional structure. Enantiomeric proteins are expected to display reciprocal chiral specificity in all aspects of their biochemical interactions.

Anticoagulant plasma polymer-modified substrate

Abstract: The present invention provides a method for surface modifying a substrate, such as an implant, to provide the substrate with anticoagulant activity and resistance to the deposition of plasma proteins; and the resulting substrate. The surface of the substrate is modified by first depositing a film of a plasma polymer such as plasma polymerized N-vinyl-2-pyrrolidone or allyl alochol on the surface of the substrate. Optionally, a neutral hydrophilic spacer molecule is attached to the plasma polymer. An anticoagulant, such as heparin, is attatched either directly to the substrate or indirectly to the substrate via attatchment to the spacer molecule, when such spacer molecule is present.

Steady-state kinetic mechanism of Ras farnesyl:protein transferase.

Abstract: The steady-state kinetic mechanism of bovine brain farnesy1:protein transferase (FPTase) has been determined using a series of initial velocity studies, including both dead-end substrate and product inhibitor experiments Reciprocal plots of the initial velocity data intersected on the l/(s) axis, indicating that a ternary complex forms (sequential mechanism) and suggesting that the binding of one substrate does not affect the binding of the other The order of substrate addition was probed by determining the patterns of dead-end substrate and product inhibition Two nonhydrolyzable analogues of farnesyl diphosphate, (a-hydroxyfamesy1)phosphonic acid (1) and ( ( (farnesylmethyl)hydroxyphosphinyl)methyl) phosphonic acid (2), were both shown to be competitive inhibitors of farnesyl diphosphate and noncompetitive inhibitors of Ras-CVLS Four nonsubstrate tetrapeptides, CV(D-L)S, CVLS-NH2, N-acetyl-L-penicillamine-VIM, and CIFM, were all shown to be noncompetitive inhibitors of famesyl diphosphate and competitive inhibitors of Ras-CVLS These data are consistent with random order of substrate addition Product inhibition patterns corroborated the results found with the dead-end substrate inhibitors We conclude that bovine brain FPTase proceeds through a random order sequential mechanism Determination of steady-state parameters for several physiological Ras-CaaX variants showed that amino acid changes affected the values of KM, but not those of kcat, suggesting that the catalytic efficiencies (kat/&) of Ras-CaaX substrates depend largely upon their relative binding affinity for FPTase

Substrate inhibition of acetylcholinesterase: residues affecting signal transduction from the surface to the catalytic center.

Abstract: Amino acids located within and around the 'active site gorge' of human acetylcholinesterase (AChE) were substituted. Replacement of W86 yielded inactive enzyme molecules, consistent with its proposed involvement in binding of the choline moiety in the active center. A decrease in affinity to propidium and a concomitant loss of substrate inhibition was observed in D74G, D74N, D74K and W286A mutants, supporting the idea that the site for substrate inhibition and the peripheral anionic site overlap. Mutations of amino acids neighboring the active center (E202, Y337 and F338) resulted in a decrease in the catalytic and the apparent bimolecular rate constants. A decrease in affinity to edrophonium was observed in D74, E202, Y337 and to a lesser extent in F338 and Y341 mutants. E202, Y337 and Y341 mutants were not inhibited efficiently by high substrate concentrations. We propose that binding of acetylcholine, on the surface of AChE, may trigger sequence of conformational changes extending from the peripheral anionic site through W286 to D74, at the entrance of the 'gorge', and down to the catalytic center (through Y341 to F338 and Y337). These changes, especially in Y337, could block the entrance/exit of the catalytic center and reduce the catalytic efficiency of AChE.

Colloidal gold as a biocompatible immobilization matrix suitable for the fabrication of enzyme electrodes by electrodeposition

Abstract: Glucose oxidase, horseradish peroxidase, xanthine oxidase, and carbonic anhydrase have been adsorbed to colloidal gold sols with good retention of enzymatic activity. Adsorption of xanthine oxidase on colloidal gold did not result in a change in enzymatic activity as determined by active site titration with the stoichiometric inhibitor pterin aldehyde and by measurement of the apparent Michaelis constant (K′M). Gold sols with adsorbed glucose oxidase, horseradish peroxidase, and xanthine oxidase have also been electrodeposited onto conducting matrices (platinum gauze and/or glassy carbon) to make enzyme electrodes. These electrodes retained enzymatic activity and, more importantly, gave an electrochemical response to the enzyme substrate in the presence of an appropriate electron transfer mediator. Our results demonstrate the utility of colloidal gold as a biocompatible enzyme imobilization matrix suitable for the fabrication of enzyme electrodes. © 1992 John Wiley & Sons, Inc.

Biosensor utilizing enzyme and a method for producing the same

Abstract: The biosensor of the present invention quantifies a substrate contained in a sample liquid by reducing electron acceptors using electron generated in a reaction of the substrate to enzyme and then by measuring the reduced amount of the electron acceptors electrochemically. The biosensor has an electrical insulating substrate, an electrode system including at least a working electrode and a counter electrode, a reaction layer including the enzyme provided on the electrode system and a hydrogen ion concentration control layer, and the reaction layer is in contact with the electrode system. According to the present invention, the hydrogen ion concentration in the sample liquid can be made most appropriate in accordance with the type of the enzyme contained in the reaction layer, without the adjustment of the hydrogen ion concentration in the sample liquid beforehand. Thus, the specific substrate contained in the sample liquid can be easily quantified with accuracy and speed.

Evidence for a sialosyl cation transition-state complex in the reaction of sialidase from influenza virus.

Abstract: The enzyme mechanism of sialidase from influenza virus has been investigated by kinetic isotope methods, NMR, and a molecular dynamics simulation of the enzyme-substrate complex. Comparison of the reaction rates obtained with the synthetic substrate 4-methylumbelliferyl-N-acetyl-α-D-neuraminic acid and the [3,3-2H]-substituted substrate revealed β-deuterium isotope effects for V/Km ranging over 1.09–1.15 in the pH range 6.0–9.5, whereas the effects observed for V in this pH range increased from 0.979 to 1.07. In D2O, βDV/Km was slightly increased by 2% and 5% at pD 6.0 and 9.5 respectively, while βDV was unchanged. Solvent isotope effects of 1.74 were obtained for both βDV/Km and βDV at pD 9.5, with βDV/Km decreasing and βDV remaining constant at acidic pD. 1H-NMR experiments confirmed that the initial product of the reaction is the α-anomer of N-acetyl-D-neuraminic acid. Molecular dynamics studies identified a water molecule in the crystal structure of the sialidase-N-acetyl-D-neuraminic acid complex which is hydrogen-bonded to Asp151 and is available to act as a proton donor source in the enzyme reaction. The results of this study lead us to propose a mechanism for the solvent-mediated hydrolysis of substrate by sialidase that requires the formation of an endocyclic sialosyl cation transition-state intermediate.

Diode-like behaviour of a mitochondrial electron-transport enzyme.

Abstract: In mitochondria, electrons derived from the oxidation of succinate by the tricarboxylic acid cycle enzyme succinate-ubiquinone oxido-reductase are transferred directly to the quinone pool. Here we provide evidence that the soluble form of this enzyme (succinate dehydrogenase) behaves as a diode that essentially allows electron flow in one direction only. The gating effect is observed when electrons are exchanged rapidly and directly between fully active succinate dehydrogenase and a graphite electrode. Turnover is therefore measured under conditions of continuously variable electrochemical potential. The otherwise rapid and efficient reduction of fumarate (the reverse reaction) is severely retarded as the driving force (overpotential) is increased. Such behaviour can arise if a rate-limiting chemical step like substrate binding or product release depends on the oxidation state of a redox group on the enzyme. The observation provides, for a biological electron-transport system, a simple demonstration of directionality that is enforced by kinetics as opposed to that which is assumed from thermodynamics.

Method for producing high quality thin layer films on substrates

Abstract: A method for producing high quality, thin layer films of inorganic compounds upon the surface of a substrate is disclosed The method involves condensing a mixture of preselected molecular precursors on the surface of a substrate and subsequently inducing the formation of reactive species using high energy photon or charged particle irradiation The reactive species react with one another to produce a film of the desired compound upon the surface of the substrate

Effects of nonionic surfactant on enzymatic hydrolysis of used newspaper

Abstract: Effects of five types of nonionic surfactant having a polyoxyethylene glycol (POG) group on enzymatic hydrolysis of used newspaper were studied. The surfactants examined in this work always enhanced the saccharification. The optimum surfactant concentration was 0.05% (wt/substrate wt) in the case of POG(21) sorbitane oleic ester. Among the surfactants, POG phenyl ether types showed the highest enhancement effect, for example, with two times higher conversion at 80 h than that without surfactant. Using POG nonylphenyl ether series, the effects of surfactant were considered from the point of the HLB (hydrophile-lypophile balance) value. The GFC (gel filtration chromatography) analysis of free enzyme quantity were also done to study the effect of surfactant on enzyme adsorption onto substrate. As the HLB value increased, the free enzyme quantity and the conversion both increased. It appears that surfactants help the enzyme to desorb from the binding site on the substrate surface after the completion of saccharification at that site.

Effects of high product and substrate inhibitions on the kinetics and biomass and product yields during ethanol batch fermentation.

Abstract: In ethanol fermentation, instantaneous biomass yield of the yeast Saccharmoyces cerevisiae was found to decrease (from 0.156 to 0.026) with increase in ethanol concentration (from 0 to 107 g/L), indicating a definite relationship between biomass yield and product inhibition. A suitable model was proposed to describe this decrease which incorporates the kinetic parameters of product inhibition rather than pure empirical constants. Substrate inhibition was found to occur when substrate concentration is above 150 g/L. A similar definite relationship was observed between substrate inhibition and instantaneous biomass yield. A simple empirical model is proposed to describe the declines in specIfic growth rate and biomass yield due to substrate inhibition. It is observed that product inhibition does not have any effect on product yield whereas substrate inhibition significantly affects the product yield, reflecting a drop in overall product yield from 0.45 to 0.30 as the initial substrate concentration increases from 150 to 280 g/L. These results are expected to have a significant influence in formulating optimum fermentor design variables and in developing an effective control strategy for optimizing ethanol producitivity.

Evaluation of a new reagent for covalent attachment of polyethylene glycol to proteins

Abstract: Methoxypolyethylene glycol of molecular weight 5000 was converted to a reactive succinimidyl carbonate form (SC-PEG). The usefulness of this new polymeric reagent for the covalent attachment of polyethylene glycol to proteins was evaluated. SC-PEG was found to be sufficiently reactive to produce extensively modified proteins under mild conditions within 30 min, showing the highest reactivity around pH 9.3. The commonly used succinimidyl succinate derivative of methoxypolyethylene glycol (SS-PEG) served as a reference standard to which the new reagent was compared. The stability of the polymer-protein linkages, studied on a series of PEG-modified bovine serum albumins, provided the single most important difference between the two activated polymers. Urethane-linked PEG-proteins obtained through the use of SC-PEG showed considerably higher chemical stability than SS-PEG-derived conjugates. The measured rate constants of aminolysis (using N alpha-acetyllysine) and hydrolysis showed that SC-PEG is slightly less reactive yet more selective of the two reagents. Hydrolysis of the active groups on SC-PEG was on average twofold slower than that on SS-PEG. The differences in the rates of aminolysis were even smaller than those in hydrolysis. PEG-trypsin conjugates produced by both activated polymers showed similar properties: they had no proteolytic activity, well-preserved esterolytic activity, and enhanced activity toward p-nitroanilide substrates. Michaelis-Menten constants of the modified enzymes were determined using N alpha-benzyloxycarbonyl-L-arginine p-nitroanilide. These measurements indicated that the attachment of PEG to trypsin caused an increase in both the rate of turnover of the substrate and its affinity toward the modified enzymes. Through a series of experiments involving the appropriate polymeric and low-molecular-weight model compounds, it was demonstrated that these increases in amidolytic activity were unrelated to tyrosyl residues acylation by either one of the activated polymers.

Isolation and amino acid sequence of a glutamic acid specific endopeptidase from Bacillus licheniformis.

Abstract: An endopeptidase cleaving specifically at the carboxyl side of acidic amino acid residues, preferentially at glutamic acid, has been isolated from a commercial extract obtained by fermentation with Bacillus licheniformis. Using ion-exchange chromatography and affinity chromatography on bacitracin-Sepharose, it was possible, from 100 ml commercial extract, to isolate 100 mg homogeneous enzyme in a yield of 50%. It is the first description of a large-scale isolation of a Glu/Asp-specific enzyme. The preparation was essentially free of contaminating activities. The isolated enzyme consists of one peptide chain of 222 amino acid residues and has a calculated molecular mass of 23 589 Da. The determined amino acid sequence shows similarity to the Glu/Asp-specific enzymes previously isolated from Staphylococcus aureus V8, Actinomyces sp. and Streptomyces thermovulgaris. The substrate preference of the enzyme has been investigated. Although non-specific cleavages were observed after prolonged hydrolysis at high enzyme concentrations the enzyme appears to be essentially specific for Glu-Xaa and Asp-Xaa, with strong preference for the former. The isolated enzyme exhibits a bell-shaped pH/activity profile with an optimum at pH 7.5–8.0. The activity is adversely affected by high ionic strength and beneficially affected by the inclusion of calcium ions in the assay medium. The enzyme is completely inhibited by diisopropylfluorophosphate, suggesting that it is a serine endopeptidase. It is partially inhibited by EDTA.

Simulation of the gas‐phase processes in remote‐plasma‐activated chemical‐vapor deposition of silicon dielectrics using rare gas–silane‐ammonia mixtures

Abstract: Remote‐plasma‐activated chemical‐vapor deposition (RPACVD) is a method whereby thin films are deposited with the substrate located out of the plasma zone. The lower rate of energetic ion and photon bombardment in RPACVD compared to conventional direct‐plasma‐enhanced chemical‐vapor deposition (DPECVD) reduces damage to the substrate. The use of RPACVD also enables one to more carefully tailor the flux of radicals to the substrate compared to DPECVD. This selectivity results from both physically isolating the substrate from undesirable radicals and limiting the variety of chemical pathways that produce radicals. A model for RPACVD is described and results from the model are discussed in the context of comparing gas mixtures and geometries in which this selectivity may be achieved. The chemistries investigated are Rg/SiH4 (Rg=Ar, He) for deposition of Si and Rg/NH3/SiH4 (Rg=Ar, He) for deposition of Si3N4. It is found that the selectivity in producing radicals that can be obtained by excitation transfer from excited states of rare gases is easily compromised by reactor configurations that allow injected gases to penetrate into the plasma zone.

Substrate preferences of glutamic-acid-specific endopeptidases assessed by synthetic peptide substrates based on intramolecular fluorescence quenching.

Abstract: The substrate preferences of the easily available Glu/Asp-specific enzymes from Staphyllococcus aureus (V8), Bacillus licheniformis and Streptomyces griseus have been extensively investigated using a series of synthetic peptide substrates, containing an N-terminal anthraniloyl group and a 3-nitrotyrosine close to the C-terminus, allowing the fluorimetric monitoring of substrate hydrolysis by the decrease in intramolecular quenching. All three enzymes hydrolysed Glu-Xaa peptide bonds approximately 1000-fold faster than Asp-Xaa bonds and they are consequently more appropriately termed Glu-specific enzymes. The difference in kcat/Km for the hydrolysis of substrates with Glu and Asp is primarily due to a difference in kcat. The enzymes appear to hydrolyse all types of Glu-Xaa bonds, although those with Xaa as Asp and, in particular, Xaa as Pro, are hydrolysed with very low rates. The influence of the nature of the amino acid residues at the substrate positions P2, P3, P4, P′1 and P′2 has been determined and it is shown that the enzyme from S. griseus exhibits the most narrow substrate preference. The results are useful in connection with fragmentation of proteins for sequencing purposes as well as for cleavage of fusion proteins.

Substrate analogue induced changes of the CO-stretching mode in the cytochrome P450cam-carbon monoxide complex.

Abstract: The CO-stretching mode of the carbon monoxide ligand in reduced cytochrome P450cam, in the absence or presence of camphor and in the presence of nine different camphor analogues, was measured at room temperature using Fourier transform infrared spectroscopy. Substrate-free cytochrome P450cam--CO reveals a broad, slightly structured band resulting from an overlap of several stretching mode signals. The multitude of the signals indicates that cytochrome P450 exists in a dynamic equilibrium of several conformational substates. Binding of camphor or camphor analogues strongly influences this equilibrium. For substrate analogues which are not able to form a hydrogen bond to the hydroxyl group of tyrosine 96, the CO-stretching band is rather broad and asymmetric. In contrast, substrate analogues with one quinone group which form a hydrogen bond to the Tyr96 OH induce a shift and a sharpening of the CO-stretching mode band. For substrate analogues with two hetero groups, the infrared spectrum is slightly asymmetric or a minor band appears. Sterical hindrance, substrate mobility, and protein flexibility finally determine the position and width of the CO-stretching mode signals.