Showing papers on "Substrate (chemistry) published in 1991"

An improved and accurate method for determining the dehydrogenase activity of soils with iodonitrotetrazolium chloride.

Abstract: Conditions for a rapid, precise [100 μg iodonitrotetrazolium chloride (INT)-formazan ml-1 assay mixture], and easily reproducible assay of potential soil dehydrogenase activity are described, using 2(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (iodonitrotetrazolium chloride, INT) as the substrate. Reduced iodonitrotetrazolium formazan (INTF) was measured by spectrophotometry (464 nm) after extraction with N,N-dimethylformamide and ethanol. With this method, the coloured complex formed is highly stable. The effects of pH, buffer concentration, temperature, substrate concentration, amount of soil weight, and reaction time on dehydrogenase activity were investigated. The rate of substrate hydrolysis was proportional to soil weight; the optimal INT reduction was achieved with 1 M TRIS buffer (pH 7.0) at 40 °C. It was possible to determine the biotic and abiotic substrate reduction by comparing assays of autoclaved and unsterile soil samples. Different investigations have confirmed that the intracellular enzyme is highly correlated with the microbial biomass, and indicate that this activity is suitable as an indirect parameter of microbial biomass, measurement.

An enzyme capable of degrading cellulose or hemicellulose.

Abstract: A cellulose- or hemicellulose-degrading enzyme which is derivable from a fungus other than Trichoderma or Phanerochaete, and which comprises a carbohydrate binding domain homologous to a terminal A region of Trichoderma reesei cellulases, which carbohydrate binding domain comprises amino acid sequence (α) or a subsequence thereof capable of effecting binding of the enzyme to an insoluble cellulosic or hemicellulosic substrate.

Crystal structures of cytochrome P-450CAM complexed with camphane, thiocamphor, and adamantane: factors controlling P-450 substrate hydroxylation

Abstract: X-ray crystal structures have been determined for complexes of cytochrome P-450CAM with the substrates camphane, adamantane, and thiocamphor. Unlike the natural substrate camphor, which hydrogen bonds to Tyr96 and is metabolized to a single product, camphane, adamantane and thiocamphor do not hydrogen bond to the enzyme and all are hydroxylated at multiple positions. Evidently the lack of a substrate-enzyme hydrogen bond allows substrates greater mobility in the active site, explaining this lower regiospecificity of metabolism as well as the inability of these substrates to displace the distal ligand to the heme iron. Tyr96 is a ligand, via its carbonyl oxygen atom, to a cation that is thought to stabilize the camphor-P-450CAM complex [Poulos, T. L., Finzel, B. C., & Howard, A. J. (1987) J. Mol. Biol. 195, 687-700]. The occupancy and temperature factor of the cationic site are lower and higher, respectively, in the presence of the non-hydrogen-bonding substrates investigated here than in the presence of camphor, underscoring the relationship between cation and substrate binding. Thiocamphor gave the most unexpected orientation in the active site of any of the substrates we have investigated to date. The orientation of thiocamphor is quite different from that of camphor. That is, carbons 5 and 6, at which thiocamphor is primarily hydroxylated [Atkins, W. M., & Sligar, S. G. (1988) J. Biol. Chem. 263, 18842-18849], are positioned near Tyr96 rather than near the heme iron. Therefore, the crystallographically observed thiocamphor-P-450CAM structure may correspond to a nonproductive complex. Disordered solvent has been identified in the active site in the presence of uncoupling substrates that channel reducing equivalents away from substrate hydroxylation toward hydrogen peroxide and/or "excess" water production. A buried solvent molecule has also been identified, which may promote uncoupling by moving from an internal location to the active site in the presence of highly mobile substrates.

Catalytic properties of an Escherichia coli formate dehydrogenase mutant in which sulfur replaces selenium

Abstract: Formate dehydrogenase H of Escherichia coli contains selenocysteine as an integral amino acid. We have purified a mutant form of the enzyme in which cysteine replaces selenocysteine. To elucidate the essential catalytic role of selenocysteine, kinetic and physical properties of the mutant enzyme were compared with those of wild type. The mutant and wild-type enzymes displayed similar pH dependencies with respect to activity and stability, although the mutant enzyme profiles were slightly shifted to more alkaline pH. Both enzymes were inactivated by reaction with iodoacetamide; however, addition of the substrate, formate, was necessary to render the enzymes susceptible to alkylation. Alkylation-induced inactivation was highly dependent on pH, with each enzyme displaying an alkylation vs. pH profile suggestive of an essential selenol or thiol. Both forms of the enzyme use a ping-pong bi-bi kinetic mechanism. The mutant enzyme binds formate with greater affinity than does the wild-type enzyme, as shown by reduced values of Km and Kd. However, the mutant enzyme has a turnover number which is more than two orders of magnitude lower than that of the native selenium-containing enzyme. The lower turnover number results from a diminished reaction rate for the initial step of the overall reaction, as found in kinetic analyses that employed the alternative substrate deuterioformate. These results indicate that the selenium of formate dehydrogenase H is directly involved in formate oxidation. The observed differences in kinetic properties may help explain the evolutionary conservation of selenocysteine at the enzyme's active site.

Structure of the triosephosphate isomerase-phosphoglycolohydroxamate complex: an analogue of the intermediate on the reaction pathway.

Abstract: The glycolytic enzyme triosephosphate isomerase (TIM) catalyzes the interconversion of the three-carbon sugars dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate (GAP) at a rate limited by the diffusion of substrate to the enzyme. We have solved the three-dimensional structure of TIM complexed with a reactive intermediate analogue, phosphoglycolohydroxamate (PGH), at 1.9-A resolution and have refined the structure to an R-factor of 18%. Analysis of the refined structure reveals the geometry of the active-site residues and the interactions they make with the inhibitor and, by analogy, the substrates. The structure is consistent with an acid-base mechanism in which the carboxylate of Glu-165 abstracts a proton from carbon while His-95 donates a proton to oxygen to form an enediol (or enediolate) intermediate. The conformation of the bound substrate stereoelectronically favors proton transfer from substrate carbon to the syn orbital of Glu-165. The crystal structure suggests that His-95 is neutral rather than cationic in the ground state and therefore would have to function as an imidazole acid instead of the usual imidazolium. Lys-12 is oriented so as to polarize the substrate oxygens by hydrogen bonding and/or electrostatic interaction, providing stabilization for the charged transition state. Asn-10 may play a similar role.

Production of Alkaline Protease by a New Aspergillus flavus Isolate under Solid-Substrate Fermentation Conditions for Use as a Depilation Agent.

Abstract: The production of alkaline protease by an Aspergillus flavus strain isolated in our laboratory by solid-substrate fermentation for use as a depilation agent and the influence of various factors on enzyme production are reported. The optimum conditions for maximum production were a growth temperature of 32°C, 63% substrate moisture, and a growth period of 48 h. Enrichment with corn steep liquor or Casitone increased productivity. Scaling-up experiments indicated that flask-scale results could be reproduced at 1 and 30 kg of substrate. The enzyme preparation exhibited maximum activity at both pH 7.5 and pH 9.5. The use of this enzyme as a depilation agent was confirmed by experiments in a tannery.

Enzyme engineering for nonaqueous solvents: random mutagenesis to enhance activity of subtilisin E in polar organic media.

Abstract: Enzyme activity is often dramatically reduced in polar organic solvents, even under conditions where the folded structures are stable. We have utilized random mutagenesis by polymerase chain reaction (PCR) techniques combined with screening for enhanced activity in the presence of dimethylformamide (DMF) to probe mechanisms by which improved enzymes for chemical synthesis in polar organic media might be obtained. Two amino acid substitutions which enhance subtilisin E activity in the presence of DMF, Q103R and D60N, were identified by screening on agar plates containing DMF and casein. The two substitutions are located near the substrate binding pocket or in the active site, and their effects on the catalytic efficiency kcat/KM for the hydrolysis of a peptide substrate are additive. The effects of D60N are apparent only in the presence of DMF, highlighting the importance of screening in the organic solvent. Protein engineering is an effective approach to enhancing enzyme activity in organic media: the triple mutant D60N + Q103R + N218S is 38 times more active than wild-type subtilisin E in 85% DMF. An evolutionary approach consisting of multiple steps of random mutagenesis and screening in continually higher concentrations of organic solvent should result in enzymes that are substantially more active in organic media.

Kinetic parameters for the elimination reaction catalyzed by triosephosphate isomerase and an estimation of the reaction's physiological significance

Abstract: Kinetic parameters for triosephosphate isomerase catalysis of the elimination reaction of an equilibrium mixture of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (DGAP) to form methylglyoxal and phosphate ion are reported for the enzyme from rabbit muscle. Pseudo-first-order rate constants for the disappearance of substrate (kelim) were determined for reactions at [Enzyme] much greater than [Substrate]. The second-order rate constant kEnz = 10.1 M-1 s-1 was determined from a plot of kelim against enzyme concentration. The kinetic parameters, determined from a steady-state kinetic analysis at [Substrate] much greater than [Enzyme], are kcat = 0.011 s-1, Km = 0.76 mM, and kcat/Km = 14 M-1 s-1. The estimated rate-constant ratio for partitioning of the enzyme-bound intermediate between protonation at carbon 2 and elimination, 1,000,000, is much larger than the ratio of 6.5 determined for the reaction of the enediolate phosphate in a loose complex with quinuclidinonium cation, a small buffer catalyst. There is a 10(5)-10(8)-fold decrease in the rate constant for the elimination reaction of the enediolate phosphate when this species binds to triosephosphate isomerase. The kinetic parameters for the elimination reaction catalyzed by the native triosephosphate isomerase and for the reaction catalyzed by a mutant form of the enzyme, which is missing a segment that forms hydrogen bonds with the phosphate group of substrate [Pompliano, D. L., Peyman, A., & Knowles, J. R. (1990) Biochemistry 29, 3186-3194] are similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Scanning Electrochemical Microscopy VII . Effect of Heterogeneous Electron‐Transfer Rate at the Substrate on the Tip Feedback Current

Abstract: The dependence of the SECM feedback current on finite heterogeneous electron-transfer kinetics at the substrate electrode was examined by experimental studies of the reduction of Fe(III) in 1M H 2 SO 4 at a Pt tip over a biased glassy-carbon substrate

X-ray analysis of D-xylose isomerase at 1.9 A: native enzyme in complex with substrate and with a mechanism-designed inactivator

Abstract: The structures of crystalline D-xylose isomerase (D-xylose ketol-isomerase; EC 5.3.1.5) from Streptomyces rubiginosus and of its complexes with substrate and with an active-site-directed inhibitor have been determined by x-ray diffraction techniques and refined to 1.9-A resolution. This study identifies the active site, as well as two metal-binding sites. The metal ions are important in maintaining the structure of the active-site region and one of them binds C3-O and C5-O of the substrate forming a six-membered ring. This study has revealed a very close contact between histidine and C1 of a substrate, suggesting that this is the active-site base that abstracts a proton from substrate. The mechanism-based inhibitor is a substrate analog and is turned over by the enzyme to give a product that alkylates this same histidine, reinforcing our interpretation. The changes in structure of the native enzyme, the enzyme with bound substrate, and the alkylated enzyme indicate that the mechanism involves an "open-chain" conformation of substrate and that the intermediate in the isomerization reaction is probably a cis-ene diol because the active-site histidine is correctly placed to abstract a proton from C1 or C2 of the substrate. A water molecule binds to C1O and C2O of the substrate and so may act as a proton donor or acceptor in the enolization of a ring-opened substrate.

On the importance of the support material for enzymatic synthesis in organic media. Support effects at controlled water activity.

Abstract: Enzymes were deposited on different porous support materials and these preparations were used to catalyze reactions in organic media. Reactions were carried out at specific water activities, achieved by equilibrating both the enzyme preparation and the substrate solution at the desired water activity before mixing them and thereby starting the reactions. The reaction rates obtained at the same water activity with different supports differed greatly, indicating a direct influence of the support on the enzyme. For horse liver alcohol dehydrogenase, Celite was the best support, and the reaction rate increased with increasing water activity. In the alpha-chymotrypsin-catalyzed alcoholysis of N-acetyl-L-phenylalanine ethyl ester with 1-butanol, high rates were again obtained with Celite, but with this support only about one third of the ethyl ester was converted to butyl ester, the rest was hydrolyzed. With the polyamide support, Accurel PA6, alcoholysis was the dominating reaction, and by using a low water activity (0.33), hydrolysis was completely suppressed while still maintaining a high alcoholysis activity. Controlled pore glass (CPG), derivatized with either hexyl or glucosyl groups, had quite different properties as enzyme supports. For horse liver alcohol dehydrogenase, glucose-CPG was a much better support than hexyl-CPG, and in the alpha-chymotrypsin-catalyzed reactions, glucose-CPG favored hydrolysis, and hexyl-CPG alcoholysis, at water activities exceeding 0.8. The results are discussed considering the absorption of water on the enzymes, on the supports and the solubility of water in the reaction media; all these parameters were measured separately.

CPT-11 Converting Enzyme from Rat Serum : Purification and Some Properties

Abstract: A rat serum enzyme that catalyzes the conversion of a pro-drug, 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11), to an anticancer drug, 7-ethyl-10-hydroxycamptothecin (SN-38), was purified and its properties were characterized. The enzyme was purified by column chromatography on diethylaminoethyl Toyopearl 650M, QAE-Sephadex, Sephadex G-150, Con A-Sepharose and high performance liquid chromatography with an ion-exchanger column. It was most active at pH 7.5 and was stable at pH 4-9 for 1 h at 30 degrees C. The molecular weight was estimated to be 60 and 57 kDa by gel filtration and sodium dodecylsulfate-polyacrylamide gel electrophoresis methods, respectively, and the isoelectric point was 4.6, as determined by isoelectric focusing. The Km value for CPT-11 was 0.28 microM. This enzyme was inhibited by diisopropyl phosphorofluoridate (DFP) and phenylmethanesulfonyl fluoride (PMSF) but insensitive to eserine, p-chloromercuribenzoate (PCMB) and ethylenediaminetetraacetate (EDTA). The enzyme also hydrolyzed p-nitrophenylacetate (p-NPA), a commonly used substrate for esterases, but was not active toward acetylcholine, suggesting that the enzyme is a carboxylesterase[EC 3.1.1.1]. During the hydrolyses of CPT-11 and p-NPA, an initial burst phenomenon similar to that found in the alpha-chymotrypsin-catalyzed hydrolysis of p-NPA was observed. Kinetic analysis revealed that the deacylation of the enzyme is the rate-limiting step in substrate hydrolysis. This enzyme was found to also split other ester derivatives of SN-38 besides CPT-11.

Phospholipase A2 at the bilayer interface.

Abstract: Interfacial catalysis is a necessary consequence for all enzymes that act on amphipathic substrates with a strong tendency to form aggregates in aqueous dispersions. In such cases the catalytic event occurs at the interface of the aggregated substrate, the overall turnover at the interface is processive, and it is influenced the molecular organization and dynamics of the interface. Such enzymes can access the substrate only at the interface because the concentration of solitary monomers of the substrate in the aqueous phase is very low. Moreover, the microinterface between the bound enzyme and the organized substrate not only facilitates formation of the enzyme-substrate complex, but a longer residence time of the enzyme at the substrate interface also promotes high catalytic processivity. Binding of the enzyme to the substrate interface as an additional step in the overall catalytic turnover permits adaptation of the Michaelis-Menten formalism as a basis to account for the kinetics of interfacial catalysis. As shown for the action of phospholipase A2 on bilayer vesicles, binding equilibrium has two extreme kinetic consequences. During catalysis in the scooting mode the enzyme does not leave the surface of the vesicle to which it is bound. On the other hand, in the hopping mode the absorption and desorption steps are a part of the catalytic turnover. In this minireview we elaborate on the factors that control binding of pig pancreatic phospholipase A2 to the bilayer interface. Binding of PLA2 to the interface occurs through ionic interactions and is further promoted by hydrophobic interactions which probably occur along a face of the enzyme, with a hydrophobic collar and a ring of cationic residues, through which the catalytic site is accessible to substrate molecules in the bilayer. An enzyme molecule binds to the surface occupied by about 35 lipid molecules with an apparent dissociation constant of less than 0.1 pM for the enzyme on anionic vesicles compared to 10 mM on zwitterionic vesicles. Results at hand also show that aggregation or acylation of the protein is not required for the high affinity binding or catalytic interaction at the interface.

Pectin methylesterase, metal ions and plant cell-wall extension. Hydrolysis of pectin by plant cell-wall pectin methylesterase.

Abstract: The hydrolysis of p-nitrophenyl acetate catalysed by pectin methylesterase is competitively inhibited by pectin and does not require metal ions to occur The results suggest that the activastion by metal ions may be explained by assuming that they interact with the substrate rather than with the enzyme With pectin used as substrate, metal ions are required in order to allow the hydrolysis to occur in the presence of pectin methylesterase This is explained by the existence of ‘blocks’ of carboxy groups on pectin that may trap enzyme molecules and thus prevent the enzyme reaction occurring Metal ions may interact with these negatively charged groups, thus allowing the enzyme to interact with the ester bonds to be cleaved At high concentrations, however, metal ions inhibit the enzyme reaction This is again understandable on the basis of the view that some carboxy groups must be adjacent to the ester bond to be cleaved in order to allow the reaction to proceed Indeed, if these groups are blocked by metal ions, the enzyme reaction cannot occur, and this is the reason for the apparent inhibition of the reaction by high concentrations of metal ions Methylene Blue, which may be bound to pectin, may replace metal ions in the ‘activation’ and ‘inhibition’ of the enzyme reaction A kinetic model based on these results has been proposed and fits the kinetic data very well All the available results favour the view that metal ions do not affect the reaction through a direct interaction with enzyme, but rather with pectin

Design and synthesis of new enzymes based on the lactate dehydrogenase framework.

Abstract: Analysis of the mechanism and structure of lactate dehydrogenases is summarized in a map of the catalytic pathway. Chemical probes, single tryptophan residues inserted at specific sites and a crystal structure reveal slow movements of the protein framework that discriminate between closely related small substrates. Only small and correctly charged substrates allow the protein to engulf the substrate in an internal vacuole that is isolated from solvent protons, in which water is frozen and hydride transfer is rapid. The closed vacuole is very sensitive to the size and charge of the substrate and provides discrimination between small substrates that otherwise have too few functional groups to be distinguished at a solvated protein surface. This model was tested against its ability to successfully predict the design and synthesis of new enzymes such as L-hydroxyisocaproate dehydrogenase and fully active malate dehydrogenase. Solvent friction limits the rate of forming the vacuole and thus the maximum rate of catalysis.

Chemical deposition methods using supercritical fluid solutions

Abstract: A method for depositing a film of a desired material on a substrate comprises dissolving at least one reagent in a supercritical fluid comprising at least one solvent. Either the reagent is capable of reacting with or is a precursor of a compound capable of reacting with the solvent to form the desired product, or at least one additional reagent is included in the supercritical solution and is capable of reacting with or is a precursor of a compound capable of reacting with the first reagent or with a compound derived from the first reagent to form the desired material. The supercritical solution is expanded to produce a vapor or aerosol and a chemical reaction is induced in the vapor or aerosol so that a film of the desired material resulting from the chemical reaction is deposited on the substrate surface. In an alternate embodiment, the supercritical solution containing at least one reagent is expanded to produce a vapor or aerosol which is then mixed with a gas containing at least one additional reagent. A chemical reaction is induced in the resulting mixture so that a film of the desired material is deposited.

Growth and substrate consumption of Nitrobacter agilis cells immobilized in carrageenan: part 2. Model evaluation.

Abstract: A dynamic model that predicts substrate and biomass concentration profiles across gel beads and from that the overall substrate consumption rate by the gel beads containing growing cells was evaluated with immobilized Nitrobacter agilis cells in an airlift loop reactor with oxygen as the limiting substrate. The model predictions agreed well with the observed oxygen consumption rates at three different liquid phase oxygen concentrations. Image analysis showed that 90% of the immobilized cells after 42 days of cultivation was situated in the outer shells in a film of 140 mum, while the bead radius was about 1 mm. The maximum biomass concentration in the outmost film of 56 mum was 11 kg . m(-3) gel.

Purification and properties of pentachlorophenol hydroxylase, a flavoprotein from Flavobacterium sp. strain ATCC 39723.

Abstract: A pentachlorophenol (PCP) hydroxylase which catalyzed the conversion of PCP to 2,3,5,6-tetrachlorohydroquinone and released iodide from triiodophenol in the presence of NADPH and oxygen was identified. The enzyme was purified by protamine sulfate precipitation, ammonium sulfate precipitation, hydrophobic chromatography, anion-exchange chromatography, gel filtration chromatography, and crystallization. The enzyme was a monomer with a molecular weight of 63,000. Under certain conditions, dimer and multimer conformations were also observed. The pI of the enzyme was pH 4.3. The optimal conditions for activity were a pH of 7.5 to 8.5 and a temperature of 40 degrees C. Each enzyme molecule contained one flavin adenine dinucleotide molecule. The Km for PCP was 30 microM and the Vmax was 16 mumol/min/mg of protein. The enzymatic reaction required 2 mol of NADPH per mol of halogenated substrate. On the basis of the data we present, it is likely that PCP hydroxylase is a flavoprotein monooxygenase. The addition of flavins to the reaction mixture did not stimulate the enzymatic reaction; however, we identified the photodegradation of triiodophenol and tribromophenol, but not PCP, by flavin mononucleotide or riboflavin and light. Images

Mechanism of enolase: the crystal structure of enolase-Mg2(+)-2-phosphoglycerate/phosphoenolpyruvate complex at 2.2-A resolution.

Abstract: Enolase in the presence of Mg2+ catalyzes the elimination of H2O from 2-phosphoglyceric acid (PGA) to form phosphoenolpyruvate (PEP) and the reverse reaction, the hydration of PEP to PGA. The structure of the ternary complex yeast enolase-Mg2(+)-PGA/PEP has been determined by X-ray diffraction and refined by crystallographic restrained least-squares to an R = 16.9% for those data with I/sigma (I) greater than or equal to 2 to 2.2-A resolution with a good geometry of the model. The structure indicates the substrate molecule in the active site has its hydroxyl group coordinated to the Mg2+ ion. The carboxylic group interacts with the side chains of His373 and Lys396. The phosphate group is H-bonded to the guanidinium group of Arg374. A water molecule H-bonded to the carboxylic groups of Glu168 and Glu211 is located at a 2.6-A distance from carbon-2 of the substrate in the direction of its proton. We propose that this cluster functions as the base abstracting the proton in the catalytic process. The proton is probably transferred, first to the water molecule, then to Glu168, and further to the substrate hydroxyl to form a water molecule. Some analogy is apparent between the initial stages of the enolase reverse reaction, the hydration of PEP, and the proteolytic mechanism of the metallohydrolases carboxypeptidase A and thermolysin. The substrate/product binding is accompanied by large movements of loops Ser36-His43 and Ser158-Gly162. The role of these conformational changes is not clear at this time.