Showing papers on "Substrate (chemistry) published in 1994"

Activation of CYP3A4: evidence for the simultaneous binding of two substrates in a cytochrome P450 active site.

Abstract: A unique characteristic of the CYP3A subfamily of cytochrome P450 enzymes is their ability to be activated by certain compounds. It is reported that CYP3A4-catalyzed phenanthrene metabolism is activated by 7,8-benzoflavone and that 7,8-benzoflavone serves as a substrate for CYP3A4. Kinetic analyses of these two substrates show that 7,8-benzoflavone increases the Vmax of phenanthrene metabolism without changing the Km and that phenanthrene decreases the Vmax of 7,8-benzoflavone metabolism without increasing the Km. These results suggest that both substrates (or substrate and activator) are simultaneously present in the active site. Both compounds must have access to the active oxygen, since neither phenanthrene nor 7,8-benzoflavone can competitively inhibit the other substrate. These data provide the first evidence that two different molecules can be simultaneously bound to the same P450 active site. Additionally, structure-activity relationship studies were performed with derivatives of 7,8-benzoflavone structure. The effects of 13 different compounds on the regioselectivity of phenanthrene, chrysene, and benzo[a]pyrene metabolism were determined. Of the 13 compounds studied, 6 were activators, 2 were partial activators, and 5 were inhibitors. Analyses of the data suggest that (1) naphthalene substituted with a ketone in the 2-position can activate 3A4 and (2) the presence of an activator results in a narrower effective substrate binding site. Since the CYP3A enzymes are very important in drug metabolism, the possibility of activation, and autoactivation, must be considered when in vitro-in vivo correlations are made and when possible drug interactions are considered.

Synthesis of polymeric microcapsule arrays and their use for enzyme immobilization

Abstract: Current methods for immobilizing enzymes for use in bioreactors and biosensors include adsorption on or covalent attachment to a support, micro-encapsulation, and entrapment within a membrane/film or gel. The ideal immobilization method should employ mild chemical conditions, allow for large quantities of enzyme to be immobilized, provide a large surface area for enzyme-substrate contact within a small total volume, minimize barriers to mass transport of substrate and product, and provide a chemically and mechanically robust system. Here we describe a method for enzyme immobilization that satisfies all of these criteria. We have developed a template-based synthetic method that yields hollow polymeric microcapsules of uniform diameter and length. These microcapsules are arranged in a high-density array in which the individual capsules protrude from a surface like the bristles of a brush. We have developed procedures for filling these microcapsules with high concentrations of enzymes. The enzyme-loaded microcapsule arrays function as enzymatic bioreactors in both aqueous solution and organic solvents.

cAMP-dependent protein kinase: crystallographic insights into substrate recognition and phosphotransfer.

Abstract: The crystal structure of ternary and binary substrate complexes of the catalytic subunit of cAMP-dependent protein kinase has been refined at 2.2 and 2.25 A resolution, respectively. The ternary complex contains ADP and a 20-residue substrate peptide, whereas the binary complex contains the phosphorylated substrate peptide. These 2 structures were refined to crystallographic R-factors of 17.5 and 18.1%, respectively. In the ternary complex, the hydroxyl oxygen OG of the serine at the P-site is 2.7 A from the OD1 atom of Asp 166. This is the first crystallographic evidence showing the direct interaction of this invariant carboxylate with a peptide substrate, and supports the predicted role of Asp 166 as a catalytic base and as an agent to position the serine -OH for nucleophilic attack. A comparison of the substrate and inhibitor ternary complexes places the hydroxyl oxygen of the serine 2.7 A from the gamma-phosphate of ATP and supports a direct in-line mechanism for phosphotransfer. In the binary complex, the phosphate on the Ser interacts directly with the epsilon N of Lys 168, another conserved residue. In the ternary complex containing ATP and the inhibitor peptide, Lys 168 interacts electrostatically with the gamma-phosphate of ATP (Zheng J, Knighton DR, Ten Eyck LF, Karlsson R, Xuong NH, Taylor SS, Sowadski JM, 1993, Biochemistry 32:2154-2161). Thus, Lys 168 remains closely associated with the phosphate in both complexes. A comparison of this binary complex structure with the recently solved structure of the ternary complex containing ATP and inhibitor peptide also reveals that the phosphate atom traverses a distance of about 1.5 A following nucleophilic attack by serine and transfer to the peptide. No major conformational changes of active site residues are seen when the substrate and product complexes are compared, although the binary complex with the phosphopeptide reveals localized changes in conformation in the region corresponding to the glycine-rich loop. The high B-factors for this loop support the conclusion that this structural motif is a highly mobile segment of the protein.

Crystal structures of medium-chain acyl-CoA dehydrogenase from pig liver mitochondria with and without substrate

Abstract: The three-dimensional structure of medium-chain acyl-CoA dehydrogenase from pig mitochondria in the native form and that of a complex of the enzyme and a substrate (product) have been solved and refined by x-ray crystallographic methods at 2.4-A resolution to R factors of 0.172 and 0.173, respectively. The overall polypeptide folding and the quaternary structure of the tetramer are essentially unchanged upon binding of the ligand, octanoyl (octenoyl)-CoA. The ligand binds to the enzyme at the rectus (re) face of the FAD in the crevice between the two alpha-helix domains and the beta-sheet domain of the enzyme. The fatty acyl chain of the thioester substrate is buried inside of the polypeptide and the 3'-AMP moiety is close to the surface of the tetrameric enzyme molecule. The alkyl chain displaces the tightly bound water molecules found in the native enzyme and the carbonyl oxygen of the thioester interacts with the ribityl 2'-hydroxyl group of the FAD and the main-chain carbonyl oxygen of Glu-376. The C alpha--C beta of the fatty acyl moiety lies between the flavin and the gamma-carboxylate of Glu-376, supporting the role of Glu-376 as the base that abstracts the alpha proton in the alpha--beta dehydrogenation reaction catalyzed by the enzyme. Trp-166 and Met-165 are located at the sinister (si) side of the flavin ring at the surface of the enzyme, suggesting that they might be involved in the interactions with electron transferring flavoprotein. Lys-304, the prevalent mutation site found in patients with medium-chain acyl-CoA dehydrogenase deficiency, is located approximately 20 A away from the active site of the enzyme.

Separation-free sandwich enzyme immunoassays using microporous gold electrodes and self-assembled monolayer/immobilized capture antibodies

Abstract: A novel separation-free sandwich-type enzyme immunoassay for proteins is performed by designing an electrochemical detection system that enables preferential measurement of surface-bound enzyme-labeled antibody relative to the excess enzyme-labeled reagent in the bulk sample solution. In this initial model system, the assay is carried out using gold-coated microporous nylon membranes (pore size 0.2 micron) which are mounted between two chambers of a diffusion cell. The membrane serves as both a solid phase for the sandwich assay and the working electrode in the three-electrode amperometric detection system. The capture monoclonal antibody is immobilized covalently on the gold side of the membrane via a self-assembled monolayer of thioctic acid. In the separation-free sandwich assay, both model analyte protein (human chorionic gonadotropin; hCG) and alkaline phosphatase labeled anti-hCG (ALP-Ab) are incubated simultaneously with the immobilized capture anti-hCG antibody. Surface-bound ALP-Ab is spatially resolved from the excess conjugate in the bulk sample solution by introducing the enzyme substrate (4-aminophenyl phosphate) through the back side of the porous membrane. The substrate diffuses rapidly through the porous membrane where it first encounters bound ALP-Ab at the gold surface. The enzymatically generated product, aminophenol, is detected immediately by oxidation at the gold electrode (at +0.19 V vs Ag/AgCl), and the magnitude of current is directly proportional to the concentration of hCG in the sample. The response time after substrate addition is less than 1 min, although maximum response toward the analyte protein requires a sample/conjugate preincubation time of 30 min with the porous electrode.(ABSTRACT TRUNCATED AT 250 WORDS)

Covalent Binding of Pd Catalysts to Ligating Self‐Assembled Monolayer Films for Selective Electroless Metal Deposition

Abstract: A new approach for the selective electroless (EL) metallization of surfaces is described. Surfaces are modified with a chemisorbed ligand‐bearing organosilane film, and then catalyzed with an aqueous Pd(II) catalyst solution. The catalyzed substrate is then immersed in an EL metal deposition bath to complete the metallization process. The ligating surfaces are produced by molecular self‐assembly of 2‐(trimethoxysilyl)ethyl‐2‐pyridine (PYR) on silicon or silica substrates. The catalyst consists of chloride‐containing aqueous Pd(II) solutions buffered at pH 5.0 to 6.4; oligomeric chloro and/or hydroxo‐bridged Pd(II) complexes act as the catalytic species at the surface. The activity of the catalyst has been characterized and modeled as a function of solution pH, [Cl−], and time from preparation. Adhesion of the Pd(II) EL catalyst to the substrate involves covalent bond formation with the surface ligand. An average minimum Pd(II) level on the surface of ~1015 Pd atom cm2 is shown to be necessary to initiate EL metallization of the substrate with an EL Co bath. This process involves fewer steps and displays improved selectivity compared to processes that involve a conventional Pd/Sn catalyst. Fabrication of high resolution metal patterns using the new metallization chemistry in conjunction with deep UV patterning of PYR films is demonstrated.

Production of RNA by a polymerase protein encapsulated within phospholipid vesicles.

Abstract: Catalyzed polymerization reactions represent a primary anabolic activity of all cells. It can be assumed that early cells carried out such reactions, in which macromolecular catalysts were encapsulated within some type of boundary membrane. In the experiments described here, we show that a template-independent RNA polymerase (polynucleotide phosphorylase) can be encapsulated in dimyristoyl phosphatidylcholine vesicles without substrate. When the substrate adenosine diphosphate (ADP) was provided externally, long-chain RNA polymers were synthesized within the vesicles. Substrate flux was maximized by maintaining the vesicles at the phase transition temperature of the component lipid. A protease was introduced externally as an additional control. Free enzyme was inactivated under identical conditions. RNA products were visualized in situ by ethidium bromide fluorescence. The products were harvested from the liposomes, radiolabeled, and analyzed by polyacrylamide gel electrophoresis. Encapsulated catalysts represent a model for primitive cellular systems in which an RNA polymerase was entrapped within a protected microenvironment.

Apatite coated on organic polymers by biomimetic process: Improvement in its adhesion to substrate by NaOH treatment

Abstract: A dense, uniform and highly biologically active bone-like apatite layer can be formed in arbitrary thickness on any kind and shape of solid substrate surface by the following biomimetic method at ordinary temperature and pressure. First, a substrate is set in contact with particles of bioactive CaO SiO2 based glass soaked in a simulated body fluid (SBF) with inorganic ion concentrations nearly equal to those of human blood plasma. Second, the substrate is soaked in another solution with ion concentrations 1.5 times those of SBF (1.5 SBF). In the present study, organic polymer substrates treated with 5 M NaOH solution were subjected to the above mentioned biomimetic process. The induction periods for the apatite nucleation on polyethyleneterephthalate (PET), polymethylmethacrylate (PMMA), polyamide 6 (PA6), and polyethersulfone (PESF) substrates were reduced from 24 to 12 h with the NaOH treatment. The adhesive strength of the formed apatite layer were increased from 3.5 to 8.6 MPa, from 1.1 to 3.4 MPa, and from 0.6 to 5.3 MPa with the NaOH treatment, for PET, PMMA, and PA 6, respectively. It was assumed that highly polar groups, such as carboxyl and sulfinyl ones formed by the hydrolysis of an ester group on PET and PMMA and of an amide group on PA 6, or of a sulfonyl group on PESF with the NaOH treatment, attached a large number of hydrated silica dissolved from the glass particles, to accelerate the apatite nucleation, and also to form a strong bond with the apatite. The apatite-organic polymer composites thus obtained are expected to be useful as bone-repairing as well as soft tissue-repairing materials.

Converting Trypsin to Chymotrypsin: Residue 172 Is a Substrate Specificity Determinant

Abstract: Trypsin and chymotrypsin have very similar tertiary structures, yet very different substrate specificities. Recent site-directed mutagenesis studies have shown that mutation of the residues of the substrate binding pocket of trypsin to the analogous residues of chymotrypsin does not convert trypsin into a protease with chymotrypsin-like specificity. However, chymotrypsin-like substrate specificity is attained when two surface loops are changed to the analogous residues of chymotrypsin, in conjunction with the changes in the S1 binding site [Hedstrom, L., Szilagyi, L., & Rutter, W. J. (1992) Science 255, 1249-1253). This mutant enzyme, Tr-->Ch[S1+L1+L2], is improved to a protease with 2-15% of the activity of chymotrypsin by the mutation of Tyr172 to Trp. Residue 172 interacts synergistically with the residues of the substrate binding pocket and the loops to determine substrate specificity. Further, these trypsin mutants demonstrate that substrate specificity is determined by the rate of catalytic processing rather than by substrate binding.

Adsorption and synergism of cellobiohydrolase I and II of Trichoderma reesei during hydrolysis of microcrystalline cellulose.

Abstract: Hydrolysis of microcrystalline cellulose (Avicel) by cellobiohydrolase I and II (CBH I and II) from Trichoderma reesei has been studied. Adsorption and synergism of the enzymes were investigated. Experiments were performed at different temperatures and enzyme/substrate ratios using CBH I and CBH II alone and in reconstituted equimolar mixtures. Fast protein liquid chromatography (FPLC) analysis was found to be an accurate and reproducible method to follow the enzyme adsorption. A linear correlation was found between the conversion and the amount of adsorbed enzyme when Avicel was hydrolyzed by increasing amounts of CBH I and/or CBH II. CBH I had lower specific activity compared to CBH II although, over a wide concentration range, more CBH I was adsorbed than CBH II. Synergism between the cellobiohydrolases during hydrolysis of the amorphous fraction of Avicel showed a maximum as a function of total enzyme concentration. Synergism measured as a function of bound enzyme showed a continuous increase, which indicates that by decreasing the distance between the two enzymes the synergism is enhanced. The adsorption process for both enzymes was slow. Depending on the enzyme/substrate ratio it took 30--90 min to reach 95% of the equilibrium binding. The amount of bound enzyme decreased withmore » increasing temperature. The two enzymes compete for the adsorption sites but also bind to specific sites. Stronger competition for adsorption sites was shown by CBH I.« less

Device for rinsing and drying substrate

Abstract: Deionized water is supplied into a rinsing bath from its bottom portion and overflows from the upper portion of the rinsing bath to form an upflow of deionized water in the rinsing bath. A substrate is rinsed by being immersed in the upflow of deionized water. After rinsing, the substrate is removed from deionized water, and at this tim the surroundings of the substrate are supplied with vapor of an organic solvent soluble in water which serves to lower surface tension of deionized water to the substrate. Thereafter, the substrate is dried in a sealed chamber that is evacuated and the surroundings of the substrate are reduced in pressure. As a result, during drying the substrate surface after rinsing same with deionized water, it is possible to reduce adhesion of particles to the substrate surface, and to dry the substrate surface rapidly without heating the substrate.

Method of forming a plasma polymerized film

Abstract: A method of preparing a coated substrate is disclosed. The substrate is coated with a plasma generated polymer containing Si, O, C and H in specific atom ratio wherein the polymer also contains certain functional groups. A power density of about 106 to about 108 J/Kg is employed in the plasma polymerization process.

Enzymatic hydrolysis of whey proteins. I: Kinetic models

Abstract: We have studied the enzymatic hydrolysis of whey proteins at pH 8 and50°C with two proteases of bacterial origin, MKC Protease 660 L, and one of animal origin, PEM 2500 S. Our results show that a greater degree of hydrolysis is achieved under the same experimental conditions with the bacterial proteases than with the animal one. In our interpretation of the results we propose a mechanism in which the hydrolytic reaction is a zero-order one for the substrate, and the enzyme denaturalizes simultaneously via a second-order kinetic process due to free enzyme attacking enzyme bound to the substrate. Our results also indicate that there is an irreversible serine-protease inhibitor in whey proteins. © 1994 John Wiley & Sons, Inc.

Amperometric biosensors for detection of L‐ and D‐amino acids based on coimmobilized peroxidase and L‐ and D‐amino acid oxidases in carbon paste electrodes

Abstract: An apparent direct electron transfer between various electrode materials and peroxidases immobilized on the surface of the electrode has been reported in the last few years. An electrocatalytic reduction of hydrogen peroxide promoted by the immobilized peroxidase starts already at about +600 mV vs SCE at neutral pH. The efficiency of the electrocatalytic current increases as the applied potential is made more negative and starts to level off at about -200 mV vs SCE. Amperometric biosensors for hydrogen peroxide can be constructed with these kind of peroxidase modified electrodes. By co-immobilizing a hydrogen peroxide producing oxidase with the peroxidase, amperometric biosensors can be made responding for the substrate of the oxidase within a potential range essentially free of interfering electrochemical reactions. Sensors for L- and D-amino acids are shown with preliminary results obtained for L- and D-phenylalanine, respectively.

Mechanism of inositol monophosphatase, the putative target of lithium therapy

Abstract: myo-Inositol monophosphatase (myo-inositol-1-phosphate phosphohydrolase, EC 3.1.3.25) is an attractive target for mechanistic investigation due to its critical role in the phosphatidylinositol signaling pathway and the possible relevance of its inhibition by Li+ to manic depression therapy. The x-ray crystallographic structure of human inositol monophosphatase in the presence of the inhibitory metal Gd3+ showed only one metal bound per active site, whereas in the presence of Mn2+, three ions were present with one being displaced upon phosphate binding. We report here modeling, kinetic, and mutagenesis studies on the enzyme, which reveal the requirement for two metal ions in the catalytic mechanism. Activity titration curves with Zn2+ or Mn2+ in the presence or absence of Mg2+ are consistent with a two-metal mechanism. Modeling studies based on the various x-ray crystallographic structures (including those with Gd3+ and substrate bound) further support a two-metal mechanism and define the positions of the two metal ions relative to substrate. While the first metal ion may activate water for nucleophilic attack, a second metal ion, coordinated by three aspartate residues, appears to act as a Lewis acid, stabilizing the leaving inositol oxyanion. In this model, the 6-OH group of substrate acts as a ligand for this second metal ion, consistent with the reduced catalytic activity observed with substrate analogues lacking the 6-OH. Evidence from Tb3+ fluorescence quenching and the two-metal kinetic titration curves suggests that Li+ binds at the site of this second metal ion.

Glucose oxidation at ruthenium dioxide based electrodes

Abstract: The oxidation of glucose at RuO2–carbon paste composite electrodes in alkaline solution was examined. It is proposed that the catalytically active species are surface-bound oxyruthenium groups, and that the latter mediate substrate oxidation via a process of cyclic heterogeneous redox catalysis. The latter process can be described in terms of a Michaelis–Menten mechanism involving the formation of a substrate–catalyst complex which subsequently decomposes to form product and pre-catalyst; the latter can subsequently be regenerated electrochemically. Hence the oxyruthenium surface groups operate as inorganic enzyme analogues.

Enantioselective hydrogenation of α-ketoesters over Pt/alumina modified with cinchonidine: theoretical investigation of the substrate-modifier interaction

Abstract: Enantio-differentiation in the asymmetric hydrogenation of α-ketoesters to α-hydroxyesters over platinum catalysts modified with cinchona-alkaloid modifiers occurs through interaction of the ketoester with the cinchona modifier. The structure of the probable transition complex has been calculated for the system methyl pyruvate (substrate) cinchonidine (modifier) using molecular mechanics and quantum chemistry techniques at both ab initio and semiempirical levels. The calculations suggest that protonated cinchonidine is energetically more likely to interact with the substrate and that the crucial interaction occurs via hydrogen bonding of the quinuclidine nitrogen and the oxygen of the α-carbonyl moiety of methyl pyruvate. In this complex the methyl pyruvate is transformed into a half-hydrogenated species which is adsorbed on the platinum surface and on hydrogenation yields the product methyll actate. Theoretical studies indicate that adsorption of the complex leading to (R) -methyl lactate is energetically more favourable than that of the corresponding complex which yields (S) -methyl lactate, which may be the key for the enantio-differentiation.

Crystal-structures of wild-type p-hydroxybenzoate hydroxylase complexed with 4-aminobenzoate, 2,4-dihydroxybenzoate, and 2-hydroxy-4-aminobenzoate and of the tyr222ala mutant complexed with 2-hydroxy-4-aminobenzoate - evidence for a proton channel and a new binding mode of the flavin ring

Abstract: The crystal structures of wild-type p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, complexed with the substrate analogues 4-aminobenzoate, 2,4-dihydroxybenzoate, and 2-hydroxy-4-aminobenzoate have been determined at 2.3-, 2.5-, and 2.8-A resolution, respectively. In addition, the crystal structure of a Tyr222Ala mutant, complexed with 2-hydroxy-4-aminobenzoate, has been determined at 2.7-A resolution. The structures have been refined to R factors between 14.5% and 15.8% for data between 8.0 A and the high-resolution limit. The differences between these complexes and the wild-type enzyme-substrate complex are all concentrated in the active site region. Binding of substrate analogues bearing a 4-amino group (4-aminobenzoate and 2-hydroxy-4-aminobenzoate) leads to binding of a water molecule next to the active site Tyr385. As a result, a continuous hydrogen-bonding network is present between the 4-amino group of the substrate analogue and the side chain of His72. It is likely that this hydrogen-bonding network is transiently present during normal catalysis, where it may or may not function as a proton channel assisting the deprotonation of the 4-hydroxyl group of the normal substrate upon binding to the active site. Binding of substrate analogues bearing a hydroxyl group at the 2-position (2,4-dihydroxybenzoate and 2-hydroxy-4-aminobenzoate) leads to displacement of the flavin ring from the active site. The flavin is no longer in the active site (the "in" conformation) but is in the cleft leading to the active site instead (the "out" conformation). It is proposed that movement of the FAD out of the active site may provide an entrance for the substrate to enter the active site and an exit for the product to leave.

Mechanisms of Cellulases and Xylanases: A Detailed Kinetic Study of the Exo-.beta.-1,4-glycanase from Cellulomonas Fimi

Abstract: The exoglucanase/xylanase from Cellulomonas fimi (Cex) has been subjected to a detailed kinetic investigation with a range of aryl beta-D-glycoside substrates. This enzyme hydrolyzes its substrates with net retention of anomeric configuration, and thus it presumably follows a double-displacement mechanism. Values of kcat are found to be invariant with pH whereas kcat/Km is dependent upon two ionizations of pKa = 4.1 and 7.7. The substrate preference of the enzyme increases in the order glucosides < cellobiosides < xylobiosides, and kinetic studies with a range of aryl glucosides and cellobiosides have allowed construction of Broensted relationships for these substrate types. A strong dependence of both kcat (beta 1g = -1) and kcat/Km (beta 1g = -1) upon leaving group ability is observed for the glucosides, indicating that formation of the intermediate is rate-limiting. For the cellobiosides a biphasic, concave downward plot is seenj for kcat, indicating a change in rate-determining step across the series. Pre-steady-state kinetic experiments allowed construction of linear Broensted plots of log k2 and log (k2/Kd) for the cellobiosides of modest (beta 1g = -0.3) slope. These results are consistent with a double-displacement mechanism in which a glycosyl-enzyme intermediate is formed and hydrolyzed via oxocarbonium ion-like transition states. Secondary deuterium kinetic isotope effects and inactivation experiments provide further insight into transition-state structures and, in concert with beta 1g values, reveal that the presence of the distal sugar moiety in cellobiosides results in a less highly charged transition state.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and properties of double-stranded RNA-specific adenosine deaminase from calf thymus.

Abstract: A double-stranded RNA-specific adenosine deaminase, which converts adenosine to inosine, has been purified to homogeneity from calf thymus. The enzyme was purified approximately 340,000-fold by a series of column chromatography steps. The enzyme consists of a single polypeptide with a molecular mass of 116 kDa as determined by electrophoresis on a SDS/polyacrylamide gel. The native protein sediments at 4.2 s in glycerol gradients and has a Stokes radius of 42 A upon gel-filtration chromatography. This leads to an estimate of approximately 74,100 for the native molecular weight, suggesting that the enzyme exists as a monomer in solution. Enzyme activity is optimal at 0.1 M KCl and 37 degrees C. Divalent metal ions or ATP is not required for activity. The Km for double-stranded RNA substrate is approximately 7 x 10(-11) M. The Vmax is approximately 10(-9) mol of inosine produced per min per mg and the Kcat is 0.13 min-1.