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Substrate Molecule (substrate + molecule)
Selected AbstractsOn the Reactivity of C(sp3),H ,-Bonds: Oxygenation with Methyl(trifluoromethyl)dioxiraneEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 3 2008Rossella Mello Abstract The reactivity of C,H ,-bonds of a series of 2-substituted adamantanes 2 towards methyl(trifluoromethyl)dioxirane (1) shows a consistent dependence on the electron-withdrawing ability, either inductive or by resonance, of the substituent. The results are interpreted in terms of the ability of the substrate molecule to delocalize the electronic perturbation of the reacting center at the beginning of the reaction path. The model shows that the electronic demand from the reacting C,H ,-bond is transmitted along the substrate through a chain of hyperconjugative interactions, the relative intensities of which depend on the ,-bonds involved. The substrate molecule simultaneously provides positive and negative stabilizing hyperconjugative interactions to the reacting center, their balance defining the geometry of the system at the beginning of the reaction path. The model constitutes a new experimental approach to measurement of the perturbation induced by substituents with significant resonance contributions on an adjacent C,H ,-bond. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008) [source] One-Pot Multienzymatic Synthesis of 12-Ketoursodeoxycholic Acid: Subtle Cofactor Specificities Rule the Reaction Equilibria of Five Biocatalysts Working in a RowADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 9 2009Daniela Monti Abstract The hydroxysteroid dehydrogenases (HSDHs)-catalyzed one-pot enzymatic synthesis of 12-ketoursodeoxycholic acid (3,,7,-dihydroxy-12-oxo-5,-cholanoic acid), a key intermediate for the synthesis of ursodeoxycholic acid, from cholic acid has been investigated. This goal has been achieved by alternating oxidative and reductive steps in a one-pot system employing HSDHs with different cofactor specificity, namely NADH-dependent HSDHs in the oxidative step and an NADPH-dependent 7,-HSDH in the reductive one. Coupled in situ regeneration systems have been exploited not only to allow the use of catalytic amounts of the cofactors, but also to provide the necessary driving force to opposite reactions (i.e., oxidation and reduction) acting on different sites of the substrate molecule. Biocatalysts suitable for the set-up of this process have been selected and their kinetic behaviour in respect of the reactions of interest has been evaluated. Finally, the process has been studied employing the enzymes both in free and compartmentalized form. [source] Free and ATP-bound structures of Ap4A hydrolase from Aquifex aeolicus V5ACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2010Jeyaraman Jeyakanthan Asymmetric diadenosine tetraphosphate (Ap4A) hydrolases degrade the metabolite Ap4A back into ATP and AMP. The three-dimensional crystal structure of Ap4A hydrolase (16,kDa) from Aquifex aeolicus has been determined in free and ATP-bound forms at 1.8 and 1.95,Å resolution, respectively. The overall three-dimensional crystal structure of the enzyme shows an ,,,-sandwich architecture with a characteristic loop adjacent to the catalytic site of the protein molecule. The ATP molecule is bound in the primary active site and the adenine moiety of the nucleotide binds in a ring-stacking arrangement equivalent to that observed in the X-ray structure of Ap4A hydrolase from Caenorhabditis elegans. Binding of ATP in the active site induces local conformational changes which may have important implications in the mechanism of substrate recognition in this class of enzymes. Furthermore, two invariant water molecules have been identified and their possible structural and/or functional roles are discussed. In addition, modelling of the substrate molecule at the primary active site of the enzyme suggests a possible path for entry and/or exit of the substrate and/or product molecule. [source] Structure of d -3-hydroxybutyrate dehydrogenase prepared in the presence of the substrate d -3-hydroxybutyrate and NAD+ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2009Md Mominul Hoque d -3-Hydroxybutyrate dehydrogenase from Alcaligenes faecalis catalyzes the reversible conversion between d -3-hydroxybutyrate and acetoacetate. The enzyme was crystallized in the presence of the substrate d -3-hydroxybutyrate and the cofactor NAD+ at the optimum pH for the catalytic reaction. The structure, which was solved by X-ray crystallography, is isomorphous to that of the complex with the substrate analogue acetate. The product as well as the substrate molecule are accommodated well in the catalytic site. Their binding geometries suggest that the reversible reactions occur by shuttle movements of a hydrogen negative ion from the C3 atom of the substrate to the C4 atom of NAD+ and from the C4 atom of NADH to the C3 atom of the product. The reaction might be further coupled to the withdrawal of a proton from the hydroxyl group of the substrate by the ionized Tyr155 residue. These structural features strongly support the previously proposed reaction mechanism of d -3-hydroxybutyrate dehydrogenase, which was based on the acetate-bound complex structure. [source] Mutation of residues critical for benzohydroxamic acid binding to horseradish peroxidase isoenzyme CBIOPOLYMERS, Issue 5 2001Barry D. Howes Abstract Aromatic substrate binding to peroxidases is mediated through hydrophobic and hydrogen bonding interactions between residues on the distal side of the heme and the substrate molecule. The effects of perturbing these interactions are investigated by an electronic absorption and resonance Raman study of benzohydroxamic acid (BHA) binding to a series of mutants of horseradish peroxidase isoenzyme C (HRPC). In particular, the Phe179 , Ala, His42 , Glu variants and the double mutant His42 , Glu:Arg38 , Leu are studied in their ferric state at pH 7 with and without BHA. A comparison of the data with those previously reported for wild-type HRPC and other distal site mutants reaffirms that in the resting state mutation of His42 leads to an increase of 6-coordinate aquo heme forms at the expense of the 5-coordinate heme state, which is the dominant species in wild-type HRPC. The His42Glu:Arg38Leu double mutant displays an enhanced proportion of the pentacoordinate heme state, similar to the single Arg38Leu mutant. The heme spin states are insensitive to mutation of the Phe179 residue. The BHA complexes of all mutants are found to have a greater amount of unbound form compared to the wild-type HRPC complex. It is apparent from the spectral changes induced on complexation with BHA that, although Phe179 provides an important hydrophobic interaction with BHA, the hydrogen bonds formed between His42 and, in particular, Arg38 and BHA assume a more critical role in the binding of BHA to the resting state. © 2001 John Wiley & Sons, Inc. Biopolymers (Biospectroscopy) 62: 261,267, 2001 [source] Cumulative bondomers: A new concept in flux analysis from 2D [13C,1H] COSY NMR dataBIOTECHNOLOGY & BIOENGINEERING, Issue 7 2002Wouter A. van Winden Abstract A well-established way of determining metabolic fluxes is to measure 2D [13C,1H] COSY NMR spectra of components of biomass grown on uniformly 13C-labeled carbon sources. When using the entire set of measured data to simultaneously determine all fluxes in a proposed metabolic network model, the 13C-labeling distribution in all measured compounds has to be simulated. This requires very large sets of isotopomer or cumomer balances. This article introduces the new concept of bondomers; entities that only vary in the numbers and positions of C,C bonds that have remained intact since the medium substrate molecule entered the metabolism. Bondomers are shown to have many analogies to isotopomers. One of these is that bondomers can be transformed to cumulative bondomers, just like isotopomers can be transformed to cumomers. Similarly to cumomers, cumulative bondomers allow an analytical solution of the entire set of balances describing a metabolic network. The main difference is that cumulative bondomer models are considerably smaller than corresponding cumomer models. This saves computational time, allows easier identifiability analysis, and yields new insights in the information content of 2D [13C,1H] COSY NMR data. We illustrate the theoretical concepts by means of a realistic example of the glycolytic and pentose phosphate pathways. The combinations of 2D [13C,1H] COSY NMR data that allow identification of all metabolic fluxes in these pathways are analyzed, and it is found that the NMR data contain less information than was previously expected. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 731,745, 2002. [source] Effect of substrate size on immunoinhibition of amylase activity,JOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 2 2001Ilka Warshawsky Abstract Immunoinhibition assays are hypothesized to work by antibodies blocking substrate access to enzyme active sites. To test this hypothesis, the inhibition of amylase isoenzymes by monoclonal and polyclonal antisera was assessed using substrates of varying sizes: chromogenic sustrates 3, 5, or 7 glucose units in length, novel synthetic macromolecular substrates, and starch. The synthetic macromolecular substrates consisted of small oligosaccharide substrates linked to an inert polymer that conferred a large size to substrate molecules as determined by gel filtration chromatography. When substrate size increased, amylase activity could be inhibited equivalently by antibody concentrations that are 10‐fold lower. Progressively less polyclonal serum was required to inhibit amylase activity as substrate length increased from 3 to 5 to 7 glucose units and as size was increased by linkage to a polymer. Different effects of substrate size were observed with two monoclonal antibodies. One monoclonal antibody blocked amylase activity independent of substrate size, while another monoclonal antibody had little inhibitory effect except using starch as substrate. We conclude that use of larger substrates can expand the repertoire of inhibitory epitopes on enzymes and convert a noninhibitory antibody into an inhibitory one. J. Clin. Lab. Anal. 15:64–70, 2001. [source] Bivalent phenethylamines as novel dopamine transporter inhibitors: evidence for multiple substrate-binding sites in a single transporterJOURNAL OF NEUROCHEMISTRY, Issue 6 2010Kyle C. Schmitt J. Neurochem. (2010) 112, 1605,1618. Abstract Bivalent ligands , compounds incorporating two receptor-interacting moieties linked by a flexible chain , often exhibit profoundly enhanced binding affinity compared with their monovalent components, implying concurrent binding to multiple sites on the target protein. It is generally assumed that neurotransmitter sodium symporter (NSS) proteins, such as the dopamine transporter (DAT), contain a single domain responsible for recognition of substrate molecules. In this report, we show that molecules possessing two substrate-like phenylalkylamine moieties linked by a progressively longer aliphatic spacer act as progressively more potent DAT inhibitors (rather than substrates). One compound bearing two dopamine (DA)-like pharmacophoric ,heads' separated by an 8-carbon linker achieved an 82-fold gain in inhibition of [3H] 2,-carbomethoxy-3,-(4-fluorophenyl)-tropane (CFT) binding compared with DA itself; bivalent compounds with a 6-carbon linker and heterologous combinations of DA-, amphetamine- and ,-phenethylamine-like heads all resulted in considerable and comparable gains in DAT affinity. A series of short-chain bivalent-like compounds with a single N -linkage was also identified, the most potent of which displayed a 74-fold gain in binding affinity. Computational modelling of the DAT protein and docking of the two most potent bivalent (-like) ligands suggested simultaneous occupancy of two discrete substrate-binding domains. Assays with the DAT mutants W84L and D313N , previously employed by our laboratory to probe conformation-specific binding of different structural classes of DAT inhibitors , indicated a bias of the bivalent ligands for inward-facing transporters. Our results strongly indicate the existence of multiple DAT substrate-interaction sites, implying that it is possible to design novel types of DAT inhibitors based upon the ,multivalent ligand' strategy. [source] EPR study of nitroxides formed from the reaction of nitric oxide with photolyzed amidesMAGNETIC RESONANCE IN CHEMISTRY, Issue 9 2003Fan Wang Abstract Free radicals generated from UV irradiation of simple aliphatic amides in anaerobic and nitric oxide (NO)-saturated liquid mixtures or solutions gave EPR spectra of nitroxides. The application of isotopic effects to EPR spectra and the generation of radicals by transient radical attack on substrate molecules or by photolysing amine or acetoin were used to help identify photochemically produced radicals from the amides. The aliphatic amides used were formamide, acetamide and their N -methyl- or deuterium-substituted derivatives. Transient radicals used to attack the amides via hydrogen-atom abstraction were generated from the initiator AIBN or AAPH. The observation of various nitroxides indicates the reactivity of NO for trapping acyl, carbamoyl and other carbon-centered radicals. Possibly mechanistic pathways diagnosed with this trap are proposed. Copyright © 2003 John Wiley & Sons, Ltd. [source] | ||||||||||||||||||||||