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Enzyme Kinetics (enzyme + kinetics)
Selected AbstractsEnzyme kinetics and mechanism, by Paul F. Cook and W.W. ClelandPROTEIN SCIENCE, Issue 2 2008Jack F. Kirsch No abstract is available for this article. [source] The Role of Human CYP2C8 and CYP2C9 Variants in Pioglitazone Metabolism In VitroBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 6 2009Eugen Muschler The present study was conducted to further clarify the role of individual CYPs and of the CYP2C8/9 polymorphisms in the primary metabolism of pioglitazone in vitro. Pioglitazone (2,400 ,M) was incubated with isolated cytochrome P450 enzymes or human liver microsomes, some of them carrying either the CYP2C8*3/*3 genotype (and also the CYP2C9*2/*2 genotype) or the CYP2C8*1/*1 genotype (five samples each). The formation of the primary pioglitazone metabolite M-IV was monitored by HPLC. Enzyme kinetics were estimated assuming a single binding site. Mean intrinsic clearance of pioglitazone to the metabolite M-IV was highest for CYP2C8 and CYP1A2 with 58 pmol M-IV/min/nmol CYP P450/,M pioglitazone each, 53 for CYP2D6*1, 40 for CYP2C19*1, and 34 for CYP2C9*2, respectively. CYP2A6, CYP2B6, CYP2C9*1, CYP2C9*3, CYP2E1, CYP3A4 and CYP3A5 did not form quantifiable amounts of M-IV. CYP2C8*1/*1 microsomes (25 ± 4 pmol M-IV/min/mg protein/,M pioglitazone) showed lower intrinsic clearance of pioglitazone than CYP2C8*3/*3 microsomes (35 ± 9, p = 0.04). In all samples, metabolite formation showed substrate inhibition, while pioglitazone did not inhibit CYP2C8-mediated paclitaxel metabolism. CYP2C8, CYP1A2 and CYP2D6 are major CYPs forming M-IV in vitro. The higher activity of CYP2C8*3/CYP2C9*2 microsomes may result from a contribution of CYP2C9*2, or from differences in CYP2C8 expression. The evidence for substrate-specific inhibitory effects of pioglitazone on CYP2C-mediated metabolism needs to be tested in further studies. [source] Organometallic Ruthenium Inhibitors of Glutathione- S -Transferase P1-1 as Anticancer DrugsCHEMMEDCHEM, Issue 12 2007Han Ang Dr. Abstract Ruthenium,arene complexes conjugated to ethacrynic acid were prepared as part of a strategy to develop novel glutathione- S -transferase (GST) inhibitors with alternate modes of activity through the organometallic fragment, ultimately to provide targeted ruthenium-based anticancer drugs. Enzyme kinetics and electrospray mass spectrometry experiments using GST P1-1 and its cysteine-modified mutant forms revealed that the complexes are effective enzyme inhibitors, but they also rapidly inactivate the enzyme by covalent binding at Cys,47 and, to a lesser extent, Cys,101. They are highly effective against the GST Pi-positive A2780 and A2780cisR ovarian carcinoma cell lines, are among the most effective ruthenium complexes reported so far, and target ubiquitous GST Pi overexpressed in many cancers. [source] Electrophoretically mediated microanalysis for the evaluation of interspecies variation in cholinesterase metabolismELECTROPHORESIS, Issue 14 2010Joana Moura Abstract This study describes an electrophoretically mediated microanalysis method, suitable for the preclinical evaluation of the hydrolysis of ester drugs by the serum of different animals and for further characterization of human,animal correlation. Dog, cat, cow, horse, sheep, rat and human serum were diluted (25%) in the appropriate buffer and replaced the enzyme solution usually used in electrophoretically mediated microanalysis methods for the study of enzyme kinetics. They were then compared in terms of the ability to hydrolyze acetylthiocholine and butyrylthiocholine (0.25,mM) by in-capillary reaction. Human serum afforded the highest conversion rates (52% butyryltiocholine and 34% acetylthiocholine) followed by horse (31 and 35%), dog (26 and 24%), cat (22 and 14%), rat (11 and 15%) and sheep (8 and 8%). Hydrolysis by bovine serum was negligible. The method is fast (under 8,min including rinsing steps), sensitive (under 25,,M substrate could be quantified) and repeatable (RSD,2%), only requiring minute amounts of sample. [source] Simplified yet highly accurate enzyme kinetics for cases of low substrate concentrationsFEBS JOURNAL, Issue 19 2009Hanna M. Härdin Much of enzyme kinetics builds on simplifications enabled by the quasi-steady-state approximation and is highly useful when the concentration of the enzyme is much lower than that of its substrate. However, in vivo, this condition is often violated. In the present study, we show that, under conditions of realistic yet high enzyme concentrations, the quasi-steady-state approximation may readily be off by more than a factor of four when predicting concentrations. We then present a novel extension of the quasi-steady-state approximation based on the zero-derivative principle, which requires considerably less theoretical work than did previous such extensions. We show that the first-order zero-derivative principle, already describes much more accurately the true enzyme dynamics at enzyme concentrations close to the concentration of their substrates. This should be particularly relevant for enzyme kinetics where the substrate is an enzyme, such as in phosphorelay and mitogen-activated protein kinase pathways. We illustrate this for the important example of the phosphotransferase system involved in glucose uptake, metabolism and signaling. We find that this system, with a potential complexity of nine dimensions, can be understood accurately using the first-order zero-derivative principle in terms of the behavior of a single variable with all other concentrations constrained to follow that behavior. [source] Characterization of phycoviolobilin phycoerythrocyanin-,84-cystein-lyase-(isomerizing) from Mastigocladus laminosusFEBS JOURNAL, Issue 18 2002Kai-Hong Zhao Cofactor requirements and enzyme kinetics have been studied of the novel, dual-action enzyme, the isomerizing phycoviolobilin phycoerythrocyanin-,84-cystein-lyase(PVB-PEC-lyase) from Mastigocladus laminosus, which catalyses both the covalent attachment of phycocyanobilin to PecA, the apo-,-subunit of phycoerythrocyanin, and its isomerization to phycoviolobilin. Thiols and the divalent metals, Mg2+ or Mn2+, were required, and the reaction was aided by the detergent, Triton X-100. Phosphate buffer inhibits precipitation of the proteins present in the reconstitution mixture, but at the same time binds the required metal. Kinetic constants were obtained for both substrates, the chromophore (Km = 12,16 µm, depending on [PecA], kcat , 1.2 × 10,4·s,1) and the apoprotein (Km = 2.4 µm at 14 µm PCB, kcat = 0.8 × 10,4·s,1). The kinetic analysis indicated that the reconstitution reaction proceeds by a sequential mechanism. By a combination of untagged and His-tagged subunits, evidence was obtained for a complex formation between PecE and PecF (subunits of PVB-PEC-lyase), and by experiments with single subunits for the prevalent function of PecE in binding and PecF in isomerizing the chromophore. [source] Assessment of amino-acid substitutions at tryptophan 16 in ,-galactosidaseFEBS JOURNAL, Issue 5 2000Elizabeth Maranville The tryptophan residue at position 16 of coffee bean ,-galactosidase has previously been shown to be essential for enzyme activity. The potential role of this residue in the catalytic mechanism has been further studied by using site-directed mutagenesis to substitute every other amino acid for tryptophan at that site. Mutant enzymes were expressed in Pichia pastoris, a methylotrophic yeast strain, and their kinetic parameters were calculated. Only amino acids containing aromatic rings (phenylalanine and tyrosine) were able to support a significant amount of enzyme activity, but the kinetics and pH profiles of these mutants differed from wild-type. Substitution of arginine, lysine, methionine, or cysteine at position 16 allowed a small amount of enzyme activity with the optimal pH shifted towards more acidic. All other residues abolished enzyme activity. Our data support the hypothesis that tryptophan 16 is affecting the pKa of a carboxyl group at the active site that participates in catalysis. We also describe an assay for continuously measuring enzyme kinetics using fluorogenic 4-methylumbelliferyl substrates. This is useful in screening enzymes from colonies and determining the enzyme kinetics when the enzyme concentration is not known. [source] High-throughput enzyme kinetics using microarraysISRAEL JOURNAL OF CHEMISTRY, Issue 2 2007Guoxin Lu We report a microanalytical method to study enzyme kinetics. The technique involves immobilizing horseradish peroxidase on a poly-L-lysine (PLL)-coated glass slide in a microarray format, followed by applying substrate solution onto the enzyme microarray. Enzyme molecules are immobilized on the PLL-coated glass slide through electrostatic interactions, and no further modification of the enzyme or glass slide is needed. In situ detection of the products generated on the enzyme spots is made possible by monitoring the light intensity of each spot using a scientific-grade charged-coupled device (CCD). Reactions of substrate solutions of various types and concentrations can be carried out sequentially on one enzyme microarray. To account for the loss of enzyme from washing in between runs, a standard substrate solution is used for calibration. Substantially reduced amounts of substrate solution are consumed for each reaction on each enzyme spot. The Michaelis constant Km obtained by using this method is comparable to the result for homogeneous solutions. Absorbance detection allows universal monitoring, and no chemical modification of the substrate is needed. High-throughput studies of native enzyme kinetics for multiple enzymes are therefore possible in a simple, rapid, and low-cost manner. [source] Non-muscle myosin IIB helps mediate TNF cell death signaling independent of actomyosin contractility (AMC)JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2010Patrick G. Flynn Abstract Non-muscle myosin II (NM II) helps mediate survival and apoptosis in response to TNF-alpha (TNF), however, NM II's mechanism of action in these processes is not fully understood. NM II isoforms are involved in a variety of cellular processes and differences in their enzyme kinetics, localization, and activation allow NM II isoforms to have distinct functions within the same cell. The present study focused on isoform specific functions of NM IIA and IIB in mediating TNF induced apoptosis. Results show that siRNA knockdown of NM IIB, but not NM IIA, impaired caspase cleavage and nuclear condensation in response to TNF. NM II's function in promoting cell death signaling appears to be independent of actomyosin contractility (AMC) since treatment of cells with blebbistatin or cytochalasin D failed to inhibit TNF induced caspase cleavage. Immunoprecipitation studies revealed associations of NM IIB with clathrin, FADD, and caspase 8 in response to TNF suggesting a role for NM IIB in TNFR1 endocytosis and the formation of the death inducing signaling complex (DISC). These findings suggest that NM IIB promotes TNF cell death signaling in a manner independent of its force generating property. J. Cell. Biochem. 9999: 1365,1375, 2010. © 2010 Wiley-Liss, Inc. [source] Thermodynamic activity-based enzyme kinetics: Efficient tool for nonaqueous enzymologyAICHE JOURNAL, Issue 3 2001Georgina C. Sandoval Lipase-catalyzed synthesis reactions must be performed in nonaqueous media (organic solvents or solvent-free systems). The choice of the optimal solvent is usually a fastidious task that necessitates the determination of kinetic parameters in each solvent. The approach used here, to overcome the lack of a model that can predict the kinetics whatever the solvent, consists in the use of thermodynamic activities instead of concentrations of components, and assumes that activity-based kinetic parameters are the same in all solvents. This assumption is discussed, and a solution is proposed which takes into account some observed residual solvent effects. The reaction chosen to test this approach was the esterification of oleic acid with ethanol catalyzed by an immobilized lipase, Lipozyme. For this reaction, the kinetics predicted in various organic solvents and in solvent-free systems is in agreement with the experimental data. [source] Principles of pharmacodynamics and their applications in veterinary pharmacologyJOURNAL OF VETERINARY PHARMACOLOGY & THERAPEUTICS, Issue 6 2004P. LEES Pharmacodynamics (PDs) is the science of drug action on the body or on microorganisms and other parasites within or on the body. It may be studied at many organizational levels , sub-molecular, molecular, cellular, tissue/organ and whole body , using in vivo, ex vivo and in vitro methods and utilizing a wide range of techniques. A few drugs owe their PD properties to some physico-chemical property or action and, in such cases, detailed molecular drug structure plays little or no role in the response elicited. For the great majority of drugs, however, action on the body is crucially dependent on chemical structure, so that a very small change, e.g. substitution of a proton by a methyl group, can markedly alter the potency of the drug, even to the point of loss of activity. In the late 19th century and first half of the 20th century recognition of these facts by Langley, Ehrlich, Dale, Clarke and others provided the foundation for the receptor site hypothesis of drug action. According to these early ideas the drug, in order to elicit its effect, had to first combine with a specific ,target molecule' on either the cell surface or an intracellular organelle. It was soon realized that the ,right' chemical structure was required for drug,target site interaction (and the subsequent pharmacological response). In addition, from this requirement, for specificity of chemical structure requirement, developed not only the modern science of pharmacology but also that of toxicology. In relation to drug actions on microbes and parasites, for example, the early work of Ehrlich led to the introduction of molecules selectively toxic for them and relatively safe for the animal host. In the whole animal drugs may act on many target molecules in many tissues. These actions may lead to primary responses which, in turn, may induce secondary responses, that may either enhance or diminish the primary response. Therefore, it is common to investigate drug pharmacodynamics (PDs) in the first instance at molecular, cellular and tissue levels in vitro, so that the primary effects can be better understood without interference from the complexities involved in whole animal studies. When a drug, hormone or neurotransmitter combines with a target molecule, it is described as a ligand. Ligands are classified into two groups, agonists (which initiate a chain of reactions leading, usually via the release or formation of secondary messengers, to the response) and antagonists (which fail to initiate the transduction pathways but nevertheless compete with agonists for occupancy of receptor sites and thereby inhibit their actions). The parameters which characterize drug receptor interaction are affinity, efficacy, potency and sensitivity, each of which can be elucidated quantitatively for a particular drug acting on a particular receptor in a particular tissue. The most fundamental objective of PDs is to use the derived numerical values for these parameters to classify and sub-classify receptors and to compare and classify drugs on the basis of their affinity, efficacy, potency and sensitivity. This review introduces and summarizes the principles of PDs and illustrates them with examples drawn from both basic and veterinary pharmacology. Drugs acting on adrenoceptors and cardiovascular, non-steroidal anti-inflammatory and antimicrobial drugs are considered briefly to provide a foundation for subsequent reviews in this issue which deal with pharmacokinetic (PK),PD modelling and integration of these drug classes. Drug action on receptors has many features in common with enzyme kinetics and gas adsorption onto surfaces, as defined by Michaelis,Menten and Langmuir absorption equations, respectively. These and other derived equations are outlined in this review. There is, however, no single theory which adequately explains all aspects of drug,receptor interaction. The early ,occupation' and ,rate' theories each explain some, but not all, experimental observations. From these basic theories the operational model and the two-state theory have been developed. For a discussion of more advanced theories see Kenakin (1997). [source] An electrostatic network and long-range regulation of Src kinasesPROTEIN SCIENCE, Issue 11 2008Elif Ozkirimli Abstract The regulatory mechanism of Src tyrosine kinases includes conformational activation by a change in the catalytic domain tertiary structure and in domain,domain contacts between the catalytic domain and the SH2/SH3 regulatory domains. The kinase is activated when tyrosine phosphorylation occurs on the activation loop, but without phosphorylation of the C-terminal tail. Activation also occurs by allostery when contacts between the catalytic domain (CD) and the regulatory SH3 and SH2 domains are released as a result of exogenous protein binding. The aim of this work is to examine the proposed role of an electrostatic network in the conformational transition and to elucidate the molecular mechanism for long-range, allosteric conformational activation by using a combination of experimental enzyme kinetics and nonequilibrium molecular dynamics simulations. Salt dependence of the induction phase is observed in kinetic assays and supports the role of an electrostatic network in the transition. In addition, simulations provide evidence that allosteric activation involves a concerted motion coupling highly conserved residues, and spanning several nanometers from the catalytic site to the regulatory domain interface to communicate between the CD and the regulatory domains. [source] Structural study and thermodynamic characterization of inhibitor binding to lumazine synthase from Bacillus anthracisACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2010Ekaterina Morgunova The crystal structure of lumazine synthase from Bacillus anthracis was solved by molecular replacement and refined to Rcryst = 23.7% (Rfree = 28.4%) at a resolution of 3.5,Å. The structure reveals the icosahedral symmetry of the enzyme and specific features of the active site that are unique in comparison with previously determined orthologues. The application of isothermal titration calorimetry in combination with enzyme kinetics showed that three designed pyrimidine derivatives bind to lumazine synthase with micromolar dissociation constants and competitively inhibit the catalytic reaction. Structure-based modelling suggested the binding modes of the inhibitors in the active site and allowed an estimation of the possible contacts formed upon binding. The results provide a structural framework for the design of antibiotics active against B. anthracis. [source] Comparison of enzymes involved in sugar metabolism from Shang-24 (Vinifera quinguangularis) and Cabernet Sauvignon (Vinifera vinifera) at veraisonAUSTRALIAN JOURNAL OF GRAPE AND WINE RESEARCH, Issue 1 2009Q.-H. PAN Abstract Background and Aims:, Sugar is a main contributor to the quality of grape berries, but little is known about the characteristics of sugar metabolism in Chinese wild grapes. Here, enzymes related to sugar metabolism were investigated in berries of both Shang-24 (Vitis quinguangularis Rehd), a wild grape native to China, and Cabernet Sauvignon (V. vinifera L.). Methods and Results:, Analyses using high performance liquid chromatography and spectrophotometer showed that Shang-24 contained lower levels of glucose and fructose, compared with Cabernet Sauvignon, but had higher activities of enzymes related to sugar hydrolysis, particularly soluble acid invertase (SAI) and ,-amylase. Analyses of enzyme kinetics, enzyme-linked immunosorbent assay and Western blot revealed that SAI and ,-amylase in Shang-24 had low Km values and that high levels of both enzymes were present. Furthermore, a novel peptide of SAI of 105 kDa was detected in Shang-24 along with a peptide of 60 kDa that also was present in Cabernet Sauvignon. Conclusions:, It is thus suggested that biochemical characteristics of SAI and ,-amylase in Shang-24 differ from those in Cabernet Sauvignon, and the novel peptide may be related to high activity of SAI in Shang-24. Significance of the Study:, These data provide an essential basis for further study of the genetic regulation of sugar and its metabolism in grape berries. [source] Inhibitory Effects of Silibinin on Cytochrome P-450 Enzymes in Human Liver MicrosomesBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 6 2000Svane Beckmann-Knopp Silibinin, the main constituent of silymarin, a flavonoid drug from silybum marianum used in liver disease, was tested for inhibition of human cytochrome P-450 enzymes. Metabolic activities were determined in liver microsomes from two donors using selective substrates. With each substrate, incubations were carried out with and without silibinin (concentrations 3.7,300 ,M) at 37° in 0.1 M KH2PO4 buffer containing up to 3% DMSO. Metabolite concentrations were determined by HPLC or direct spectroscopy. First, silibinin IC50 values were determined for each substrate at respective KM concentrations. Silibinin had little effect (IC50>200 ,M) on the metabolism of erythromycin (CYP3A4), chlorzoxazone (CYP2E1), S(+)-mephenytoin (CYP2C19), caffeine (CYP1A2) or coumarin (CYP2A6). A moderate effect was observed for high affinity dextromethorphan metabolism (CYP2D6) in one of the microsomes samples tested only (IC50=173 ,M). Clear inhibition was found for denitronifedipine oxidation (CYP3A4; IC50=29 ,M and 46 ,M) and S(,)-warfarin 7-hydroxylation (CYP2C9; IC50=43 ,M and 45 ,M). When additional substrate concentrations were tested to assess enzyme kinetics, silibinin was a potent competitive inhibitor of dextromethorphan metabolism at the low affinity site, which is not CYP2D6 (Ki,c=2.3 ,M and 2.4 ,M). Inhibition was competitive for S(,)-warfarin 7-hydroxylation (Ki,c=18 ,M and 19 ,M) and mainly non-competitive for denitronifedipine oxidation (Ki,n=9 ,M and 12 ,M). With therapeutic silibinin peak plasma concentrations of 0.6 ,M and biliary concentrations up to 200 ,M, metabolic interactions with xenobiotics metabolised by CYP3A4 or CYP2C9 cannot be excluded. [source] Lucenz simulator: A tool for the teaching of enzyme kineticsBIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 3 2004Alan G. Clark Abstract A program has been developed that will produce simulated enzyme kinetic data suitable for inclusion in problem sets for undergraduates. Mechanisms simulated include one- and two-substrate reactions and various types of inhibition. The effects of variation of pH and temperature may be modeled, as may the effects of random errors on the experimental procedure. The graphical output is suitable for use in lecture demonstrations. [source] A hidden square-root boundary between growth rate and biomass yieldBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009Wilson W. Wong Abstract Although the theoretical value of biomass yield can be calculated from metabolic network stoichiometry, the growth rate is difficult to predict. Since the rate and yield can vary independently, no simple relationship has been discovered between these two variables. In this work, we analyzed the well-accepted enzyme kinetics and uncovered a hidden boundary for growth rate, which is determined by the square-root of three physiological parameters: biomass yield, the substrate turnover number, and the maximum synthesis rate of the turnover enzyme. Cells cannot grow faster than the square-root of the product of these parameters. This analysis is supported by experimental data and involves essentially no assumptions except (i) the cell is not undergoing a downshift transition, (ii) substrate uptake enzyme activity is proportional to its copy number. This simple boundary (not correlation) has escaped notice for many decades and suggests that the yield calculation does not predict the growth rate, but gives an upper limit for the growth rate. The relationship also explains how growth rate is affected by the yield and sheds lights on strain design for product formation. Biotechnol. Bioeng. 2009;102: 73,80. © 2008 Wiley Periodicals, Inc. [source] A comprehensive kinetic model of laccase-catalyzed oxidation of aqueous phenolBIOTECHNOLOGY PROGRESS, Issue 3 2009Selvia Kurniawati Abstract A comprehensive model was developed to describe the kinetics of the laccase-catalyzed oxidation of phenol that incorporates enzyme kinetics, enzyme inactivation, variable reaction stoichiometry between substrate and oxygen, and oxygen mass-transfer. The model was calibrated and validated against data obtained from experiments conducted in an open system, which allowed oxygen to transfer from air to the reacting mixture and phenol conversion to approach completion. Inactivation of laccase was observed over the course of the reaction and was found to be dependent on the rate of substrate transformation. A single kinetic expression was sufficient to describe laccase inactivation arising from interaction with reacting species over time. Excellent agreement was found between model predictions of phenol and oxygen concentrations and experimental data over time for a wide range of initial substrate concentrations and enzyme activities. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] A new crystal form of bovine pancreatic RNase A in complex with 2,-deoxyguanosine-5,-monophosphateACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2007Steven B. Larson The structure of bovine pancreatic RNase A has been determined in complex with 2,-deoxyguanosine-5,-monophosphate (dGMP) at 1.33,Å resolution at room temperature in a previously unreported unit cell belonging to space group P31. There are two molecules of nucleotide per enzyme molecule, one of which lies in the active-site cleft in the productive binding mode, whilst the guanine base of the other dGMP occupies the pyrimidine-specific binding site in a nonproductive mode such that it forms hydrogen bonds to the phosphate group of the first dGMP. This is the first RNase A structure containing a guanine base in the B2 binding site. Each dGMP molecule is involved in intermolecular interactions with adjacent RNase A molecules in the lattice and the two nucleotides appear to direct the formation of the crystal lattice. Because GMP may be produced during degradation of RNA, this association could represent an inhibitor complex and thereby affect the observed enzyme kinetics. [source] The Use of Biochemical and Biophysical Tools for Triage of High-Throughput Screening Hits , A Case Study with Escherichia coli Phosphopantetheine AdenylyltransferaseCHEMICAL BIOLOGY & DRUG DESIGN, Issue 5 2010J. Richard Miller High-throughput screening is utilized by pharmaceutical researchers and, increasingly, academic investigators to identify agents that act upon enzymes, receptors, and cellular processes. Screening hits include molecules that specifically bind the target and a greater number of non-specific compounds. It is necessary to ,triage' these hits to identify the subset worthy of further exploration. As part of our antibacterial drug discovery effort, we applied a suite of biochemical and biophysical tools to accelerate the triage process. We describe application of these tools to a series of 9-oxo-4,9-dihydropyrazolo[5,1-b]quinazoline-2-carboxylic acids (PQ) hits from a screen of Escherichia coli phosphopantetheine adenylyltransferase (PPAT). Initial confirmation of specific binding to phosphopantetheine adenylyltransferase was obtained using biochemical and biophysical tools, including a novel orthogonal assay, isothermal titration calorimetry, and saturation transfer difference NMR. To identify the phosphopantetheine adenylyltransferase sub-site bound by these inhibitors, two techniques were utilized: steady-state enzyme kinetics and a novel 19F NMR method in which fluorine-containing fragments that bind the ATP and/or phosphopantetheine sites serve as competitive reporter probes. These data are consistent with PQs binding the ATP sub-site. In addition to identification of a series of PPAT inhibitors, the described hit triage process is broadly applicable to other enzyme targets in which milligram quantities of purified target protein are available. [source] The Chemistry of Escapin: Identification and Quantification of the Components in the Complex Mixture Generated by an L -Amino Acid Oxidase in the Defensive Secretion of the Sea Snail Aplysia californicaCHEMISTRY - A EUROPEAN JOURNAL, Issue 7 2009Michiya Kamio Dr. Abstract A complex mixture of products in an enzymatic reaction: Aplysia californica releases amino acid oxidase and its substrate lysine in defensive secretions to produce a mixture of multiple compounds (see figure). Escapin is an L -amino acid oxidase in the ink of a marine snail, the sea hare Aplysia californica, which oxidizes L -lysine (1) to produce a mixture of chemicals which is antipredatory and antimicrobial. The goal of our study was to determine the identity and relative abundance of the constituents of this mixture, using molecules generated enzymatically with escapin and also using products of organic syntheses. We examined this mixture under the natural range of pH values for ink,from ,5 at full strength to ,8 when fully diluted in sea water. The enzymatic reaction likely forms an equilibrium mixture containing the linear form ,-keto-,-aminocaproic acid (2), the cyclic imine ,1 -piperidine-2-carboxylic acid (3), the cyclic enamine ,2 -piperidine-2-carboxylic acid (4), possibly the linear enol 6-amino-2-hydroxy-hex-2-enoic acid (7), the ,-dihydroxy acid 6-amino-2,2-dihydroxy-hexanoic acid (8), and the cyclic aminol 2-hydroxy-piperidine-2-carboxylic acid (9). Using NMR and mass spectroscopy, we show that 3 is the major component of this enzymatic product at any pH, but at more basic conditions, the equilibrium shifts to produce relatively more 4, and at acidic conditions, the equilibrium shifts to produce relatively more 2, 7, and/or 9. Studies of escapin's enzyme kinetics demonstrate that because of the high concentrations of escapin and L -lysine in the ink secretion, millimolar concentrations of 3, H2O2, and ammonia are produced, and also lower concentrations of 2, 4, 7, and 9 as a result. We also show that reactions of this mixture with H2O2 produce ,-aminovaleric acid (5) and ,-valerolactam (6), with 6 being the dominant component under the naturally acidic conditions of ink. Thus, the product of escapin's action on L -lysine contains an equilibrium mixture that is more complex than previously known for any L -amino acid oxidase. [source] | ||||||||||||||||||||||||||||