Showing papers on "Substrate (chemistry) published in 1985"
TL;DR: A Mn(II)-dependent peroxidase found in the extracellular medium of ligninolytic cultures of the white rot fungus, Phanerochaete chrysosporium, was purified and the absorption spectrum of the enzyme indicates the presence of a heme prosthetic group.
716 citations
TL;DR: Hard and softwoods were pretreated by mild acid hydrolysis and their pore size distribution determined, finding that the initial rate of Hydrolysis using cellulase from Trichoderma reesei is linearly correlated with the pore volume of the substrate accessible to a nominal diameter representative of the size of the cellulase.
Abstract: Hard and softwoods were pretreated by mild acid hydrolysis and their pore size distribution determined. Regardless of the substrate, the initial rate of hydrolysis using cellulase from Trichoderma reesei is linearly correlated with the pore volume of the substrate accessible to a nominal diameter of 51 A representative of the size of the cellulase. In contrast, crystallinity index has no relationship to the rate of hydrolysis.
535 citations
TL;DR: In this paper, the authors proposed a method to grow polycrystalline or epitaxial thin films of water-insoluble ionic or ionocovalent compounds of the CmAn type by heterogeneous chemical reaction at the solid-solution interface between adsorbed Cn+ cations and Am− anions.
376 citations
TL;DR: Although the mutant enzymes were reduced in catalytic rate, they showed enhanced substrate specificity relative to the native enzyme, achieved by the unexpected differential effects on the catalytic activity toward arginine and lysine substrates.
Abstract: A general method for modifying eukaryotic genes by site-specific mutagenesis and subsequent expression in mammalian cells was developed to study the relation between structure and function of the proteolytic enzyme trypsin. Glycine residues at positions 216 and 226 in the binding cavity of trypsin were replaced by alanine residues, resulting in three trypsin mutants. Computer graphic analysis suggested that these substitutions would differentially affect arginine and lysine substrate binding of the enzyme. Although the mutant enzymes were reduced in catalytic rate, they showed enhanced substrate specificity relative to the native enzyme. This increased specificity was achieved by the unexpected differential effects on the catalytic activity toward arginine and lysine substrates. Mutants containing alanine at position 226 exhibited an altered conformation that may be converted to a trypsin-like structure upon binding of a substrate analog.
321 citations
289 citations
TL;DR: The presence of indolylamine 2,3-dioxygenase was examined in human subjects by determining its activity with L-tryptophan as substrate.
Abstract: The presence of indolylamine 2,3-dioxygenase was examined in human subjects by determining its activity with L-tryptophan as substrate. Enzyme activity was detected in various tissues, and was relatively high in the lung, small intestine and placenta. Human indolylamine 2,3-dioxygenase, partially purified from the placenta, had an Mr of about 40 000 by gel filtration and exhibited a single pI of 6.9. The human enzyme required a reducing system, ascorbic acid and Methylene Blue, for maximal activity and was able to oxidize D-tryptophan, 5-hydroxy-L-tryptophan as well as L-tryptophan, but kinetic studies indicated that the best substrate of the enzyme was L-tryptophan.
234 citations
TL;DR: Acetylene was shown to bind to proteins which are associated with methane-oxidising activity and it is proposed that acetylene acts as a suicide substrate.
Abstract: Acetylene was shown to be an inhibitor of cell-free methane monooxygenase (MMO) activity in Methylococcus capsulatus (Bath). Inhibition was demonstrated for both the soluble and particulate forms of the enzyme and was dependent on the presence of both NADH and oxygen. Inactivation of the enzyme complex was irreversible and was due to binding of the acetylene to specific proteins of the enzyme complex. The use of radiolabelled [14C]acetylene provided a method for visualisation of the bound inhibitor: protein complex on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Acetylene was shown to bind to proteins which are associated with methane-oxidising activity and it is proposed that acetylene acts as a suicide substrate.
233 citations
TL;DR: The finding that the GM2 activatorprotein inhibits the degradation of the sulfated substrate by beta-hexosaminidases A and S indicates that the alpha subunit common to both isoenzymes might provide a binding site for the activator protein.
202 citations
TL;DR: Surprisingly, the cobra venom phospholipase normally acts poorly on phosphatidylethanolamine as substrate, but after reaction with manoalide, the enzyme is somewhat more active toward this substrate rather than being inhibited, which suggests that a lysine residue may be important in understanding the substrate specificity of phosphate A2.
170 citations
TL;DR: The cleavage of peptides by chymotrypsin and carboxypeptidase A can be interpreted satisfactorily in this way and the large rates and specificity of enzyme reactions may be due simply to elimination of the solvent.
Abstract: Since adsorption of the substrate in the active site of an enzyme can occur only if all solvent is squeezed out from between them, any reaction between them takes place in the absence of any intervening solvent--i.e., as it would in the gas phase. Recent work has shown that ionic reactions in the gas phase often differ greatly from analogous processes in solution. Therefore, current interpretations of enzyme reactions in terms of solution chemistry are misguided. The large rates and specificity of enzyme reactions may be due simply to elimination of the solvent. The cleavage of peptides by chymotrypsin and carboxypeptidase A can be interpreted satisfactorily in this way.
168 citations
TL;DR: The presence of a specific site in the Fe coordination for NO which is modified in the substrate complex, suggests that O2 binding by the extradiol dioxygenases may also occur at the Fe.
TL;DR: Phenylalanine ammonia-lyase has been shown to catalyze the elimination of ammonia from the slow alternate substrate 3-(1,4-cyclohexadienyl)alanine by an E1 cb mechanism with a carbanion intermediate, with the equations for concerted, carbanions, and carbonium ion mechanisms consistent with the same mechanism with drastically increased commitments.
Abstract: Phenylalanine ammonia-lyase has been shown to catalyze the elimination of ammonia from the slow alternate substrate 3-(1,4-cyclohexadienyl)alanine by an E1 cb mechanism with a carbanion intermediate. This conclusion resulted from comparison of 15N isotope effects with deuterated (0.9921) and unlabeled substrates (1.0047), and a deuterium isotope effect of 2.0 from dideuteration at C-3, with the equations for concerted, carbanion, and carbonium ion mechanisms. The 15N equilibrium isotope effect on the addition of the substrate to the dehydroalanine prosthetic group on the enzyme is 0.979, while the kinetic 15N isotope effect on the reverse of this step is 1.03-1.04 and the intrinsic deuterium isotope effect on proton removal is in the range 4-6. Isotope effects with phenylalanine itself are small (15N ones of 1.0021 and 1.0010 when unlabeled or 3-dideuterated and a deuterium isotope effect of 1.15) but are consistent with the same mechanism with drastically increased commitments, including a sizable external one (i.e., phenylalanine is sticky). pH profiles show that the amino group of the substrate must be unprotonated to react but that a group on the enzyme with a pK of 9 must be protonated, possibly to catalyze addition of the substrate to dehydroalanine. Incorrectly protonated enzyme-substrate complexes do not form. Equilibrium 15N isotope effects are 1.016 for the deprotonation of phenylalanine or its cyclohexadienyl analogue, 1.0192 for deprotonation of NH4+, 1.0163 for the conversion of the monoanion of phenylalanine to NH3, and 1.0138 for the conversion of the monoanion of aspartate to NH4+.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: It is proposed that the fraction of cardiac output that perfuses the udder of lactating ruminants plays a role in regulation of nutrient partitioning between milk and body tissues and investigation of the mechanism of this response should prove fruitful to understanding causes of variations of milk production in response to feed quantity and quality.
TL;DR: The results establish that P. putida is a valid whole cell model for the reductase activity of the P-450 complement in these reactions, and the chemistry observed for the enzyme parallels that of its iron(II) porphyrin active site.
Abstract: The products, stoichiometry, and kinetics of the oxidation of the enzyme cytochrome P-450 cam by five polyhalomethanes and chloronitromethane are described. The reactivity of the enzyme is compared with that of deuteroheme and with the enzyme in its native cell, Pseudomonas putida (PpG-786). In all cases, the reaction entails hydrogenolysis of the carbon-halogen bond: 2FeIIP + RCXn----2FeIIIP + RCHXn-1 (P = porphyrin or P-450 cam in vitro and in vivo). Trichloronitromethane was the fastest reacting substrate, and chloroform was the slowest. The results establish that P. putida is a valid whole cell model for the reductase activity of the P-450 complement in these reactions. The reactions of cytochrome P-450 with polyhaloalkanes proceed in a manner quite analogous to other iron(II) proteins in the G conformation. The chemistry observed for the enzyme parallels that of its iron(II) porphyrin active site. Iron-bonded carbenes are not intermediates, and hydrolytically stable iron alkyls are not products of these reactions.
TL;DR: Comparison of calcineurin and acid-catalyzed hydrolyses indicates a 1:1 correlation between the rate constants for the two processes.
Patent•
25 Nov 1985TL;DR: In this article, an electrochemical sensor is formed having a working electrode for detecting hydrogen peroxide surrounded by a cylinder portion, and with an enzymecontaining membrane at its tip.
Abstract: An electrochemical sensor is formed having a working electrode for detecting hydrogen peroxide surrounded by a cylinder portion, and with an enzymecontaining membrane at its tip. The membrane has a porous layer permeable to hydrogen peroxide between a layer containing an immobilized enzyme capable of decomposing hydrogen peroxide and a layer containing an immobilized enzyme capable of decomposing a substrate to form hydrogen peroxide. The cylinder portion is embedded in the layer containing the hydrogen peroxide decomposing enzyme and surrounds the working electrode such that the electrode is in contact with the porous layer but is not in contact with the layer containing the hydrogen peroxide decomposing enzyme. The layer containing the hydrogen peroxide forming enzyme is on a side of the porous layer opposite the electrode so as not to contact the electrode. Activity of the hydrogen peroxide decomposing enzyme is no more than one-fourth of the activity of the hydrogen peroxide forming enzyme. The electrochemical sensor reduces base line elevation after measurement action has been discontinued and measurement of a next sample is restarted.
TL;DR: Both thrombin and factor Xa are efficient enzymes toward factor VIII:C in comparison with published values of the catalytic efficiencies of several other coagulation enzymes for various substrates.
Abstract: The activation of porcine factor VIII:C by thrombin and by factor Xa was studied by a chromogenic substrate assay and by sodium dodecyl sulfate-polyacrylamide gel radioelectrophoresis of 125I-labeled factor VIII:C activation products. In the chromogenic assay, the kinetics of factor VIII:C dependent activation of factor X by factor IXa in the presence of calcium and phosphatidylserine/phosphatidylcholine vesicles were measured with N-benzoyl-L-isoleucyl-L-glutamylglycyl-L-arginine p-nitroanilide (S2222) as substrate. Substrate dependence of initial rates of the reaction at fixed factor IXa, factor VIII:C, lipid, and calcium obeyed Michaelis-Menten kinetics. At fixed factor IXa, factor X, lipid, and calcium the initial rates of the reaction varied linearly with lower factor VIII:C concentrations and plateaued at higher concentrations. The linear initial rate dependence formed the basis of a rapid, plasma-free assay of activated factor VIII:C. The activation of factor VIII:C by thrombin or factor Xa and the enzyme-independent rate of spontaneous inactivation were studied under conditions of excess enzyme. A model of the activation kinetics was developed and fit to the data by a nonlinear least-squares technique. From the model, the catalytic efficiencies (kcat/Km) of factor VIII:C activation by thrombin and factor Xa were 5.0 X 10(6) M-1 s-1 and 1.1 X 10(6) M-1 s-1, respectively. By comparison with published values of the catalytic efficiencies of several other coagulation enzymes for various substrates, both thrombin and factor Xa are efficient enzymes toward factor VIII:C.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: Lignin degradation Single‐electron transfer Cα‐Cβ bond cleavage Phanerochaete chrysosporium Peroxidase compound I
TL;DR: In this article, the fabrication process and characteristics of monolithically integrated urea and glucose FETs are described, and an insoluble membrane with enzyme entrapped in it is formed, which is used for the preparation of multifunctional and one-chip biosensors with various other pairs of immobilized enzymes.
TL;DR: In this article, the steady state operation of an amperometric enzyme is derived, and a simple diagnostic plot is derived which allows the rate limiting process to be identified, and the balance between the transport of substrate through the membrane and the enzyme and electrochemical kinetics to be determined.
TL;DR: In a 2-site binding enzyme immunoassay for alpha-1-fetoprotein (AFP) the detection limit for AFP was reduced by a factor of 2 only by the fluorimetry compared to the colorimetry with all 3 marker enzymes.
TL;DR: The kinetic mechanism of the native enzyme is similar to that established for the isolated porcine liver enzyme in that the rate-limiting step of catalysis occurs after substrate binding, and that all substrates capable of saturating the microsomal enzyme should be metabolized at a common maximal velocity.
TL;DR: The molecular and catalytic properties of the acid Tyr(P)-protein phosphatase purified from bovine heart are very similar to those of the low-molecular-weight acid phosphatases of Mr = 14,000 previously identified and purified from the cytosolic fraction of human liver, placenta, and other animal tissues.
TL;DR: In this article, the first thermal cleaning of Si is important in growing single domain GaAs and the growth temperatures of the buffer layer had to be low to get a good morphological surface.
Abstract: Good surface morphological single domain GaAs films were successfully grown on a whole area of 2-inch Si(100) substrates by molecular beam epitaxy (MBE). Si substrates were first thermally cleaned at 850°C, 100 A GaAs buffer layers were then grown at low substrate temperatures and, finally, 1.5 µm GaAs layers were grown at 600°C. It was found that the first thermal cleaning of Si is important in growing single domain GaAs and the growth temperatures of the buffer layer had to be low to get a good morphological surface.
TL;DR: The best mode of fermentation was a semicontinuous fed‐batch fermentation where one‐half of the fermented material was removed at three‐day intervals and replaced by fresh substrate, and protein production was 20% higher than in batch mode, protein productivity was maintained over 12 days, and sporulation was prevented.
Abstract: Optimal conditions for solid substrate fermentation of wheat straw with Chaetomium cellulolyticum in laboratory-scale stationary layer fermenters were developed. The best pretreatment for wheat straw was ammonia freeze explosion, followed by steam treatment, alkali treatment, and simple autoclaving. The optimal fermentation conditions were 80% (w/w) moisture content; incubation temperature of 37°C; 2% (w/w) unwashed mycelial inoculum; aeration at 0.12 L/h/g; substrate thickness of 1 to 2 cm; and duration of three days. Technical parameters for this optimized fermentation were: degree of substance utilization, 27.2%; protein yield/substrate, 0.09 g; biomass yield/bioconverted substrate, 0.40 g; degree of bioconversion of total available sugars in the substrate, 60.5%; specific efficiency of bioconversion, 70.8%; and overall efficiency of biomass production from substrate, 42.7%. Mixed culturing of Candida utilis further increased biomass production by 20%. The best mode of fermentation was a semicontinuous fed-batch fermentation where one-half of the fermented material was removed at three-day intervals and replaced by fresh substrate. In this mode, protein production was 20% higher than in batch mode, protein productivity was maintained over 12 days, and sporulation was prevented.
TL;DR: The algebraic relationship is particularly suited for use in the design of biofilm processes such as trickling filters, rotating biological contactors, anaerobic filters, and fluidized or expanded‐bed reactors.
Abstract: A simplified algebraic expression for biofilm kinetics is developed. This mathematical model assumes Monod‐type biological kinetics and diffusive mass transport. The algebraic expression relates the concentration of substrate at the biofilm surface, and the thickness of the biofilm to the substrate utilization rate. The algebraic relationship is particularly suited for use in the design of biofilm processes such as trickling filters, rotating biological contactors, anaerobic filters, and fluidized or expanded‐bed reactors.
TL;DR: It is concluded that aconitase containing a 3Fe cluster is not active in either oxidation state, as shown in the Mössbauer studies.
TL;DR: It is evident that the mutation of glutamic acid-165 to aspartic acid principally affects the free energy of the transition state(s) for the catalytic reaction itself.
Abstract: We have replaced the glutamic acid-165 at the active site of chicken triosephosphate isomerase with an aspartic acid residue using site-directed mutagenesis. Expression of the mutant protein in a strain of Escherichia coli that lacks the bacterial isomerase results in a complementation phenotype that is intermediate between strains that have no isomerase and strains that produce either the wild-type chicken enzyme or the native E. coli isomerase. The value of kcat for the purified mutant enzyme when glyceraldehyde 3-phosphate is the substrate is 1/1500th that of the wild-type enzyme, and the Km is decreased by a factor of 3.6. With dihydroxyacetone phosphate as substrate, the kcat value is 1/240th that of the wild-type enzyme, and Km is 2 times higher. The value of Ki for a competitive inhibitor, phosphoglycolate, is the same for the mutant and wild-type enzymes, at 2 X 10(-5) M. By treating the enzyme-catalyzed isomerization as a simple three step process and assuming that substrate binding is diffusion limited, it is evident that the mutation of glutamic acid-165 to aspartic acid principally affects the free energy of the transition state(s) for the catalytic reaction itself.
TL;DR: Rat liver uracil-DNA glycosylase has been purified from nuclear extracts over 3000-fold to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Abstract: Rat liver uracil-DNA glycosylase has been purified from nuclear extracts over 3000-fold to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is a monomeric protein with a polypeptide molecular weight of approximately 35 000. It has a native molecular weight of 33 000 as determined by gel filtration chromatography and a sedimentation coefficient of 2.6 S in glycerol gradients. The nuclear enzyme has an alkaline pH optimum and a pI value of 9.3. Nuclear uracil-DNA glycosylase catalyzes the release of free uracil from both single-stranded and double-stranded DNA with the former being the preferred substrate. The enzyme is unable to recognize dUTP, dUMP, or poly(dA-dT) containing a 3'-terminal uracil residue as a substrate. However, internalization of terminal uracil residues by limited chain elongation produced a substrate for the glycosylase. Another species of uracil-DNA glycosylase has been partially purified from mitochondria. This activity differs from the nuclear enzyme in that it has (i) distinctive chromatographic properties, (ii) a lower native molecular weight of 20 000 as determined by molecular sieving, (iii) a distinct NaCl inhibition profile, and (iv) a longer half-life during thermal denaturation.