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Substrate Inhibition (substrate + inhibition)
Selected AbstractsSubstrate inhibition of Pediococcus acidilactici by glucose on a waste medium.LETTERS IN APPLIED MICROBIOLOGY, Issue 5 2003Simulations, experimental results Abstract Aims: The possibility of substrate inhibition by glucose on biomass and pediocin production was studied in cultures of Pediococcus acidilactici on a residual medium. Methods and Results: Calculation of the substrate inhibition coefficient in the context of microbial growth is generally laborious, and very prone to experimental error. However, a simulation combining logistic and Monod kinetics equations demonstrates that quantitative evidence for this type of inhibition, without the possibility of misinterpretation, can be obtained through the comparison of punctual preasymptotic productions as a function of substrate concentration. Significance and Impact of the Study: It was concluded that glucose had an inhibitory effect on growth, but not on bacteriocin production. [source] Regioselective C-6 Hydrolysis of Methyl O -Benzoyl-pyranosides Catalysed by Candida Rugosa LipaseEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 10 2009Aslan Esmurziev Abstract Hydrolysis of six methyl O -benzoyl-pyranosides has been investigated using Candida rugosa lipase in dioxane/buffer mixtures. The lipase catalysed the hydrolysis of all substrates in a regiospecific manner at C-6. The rate of reaction was dependent on pyranoside structure, reaction temperature and scale, dioxane concentration and agitation speed. Starting from their C-6 O -benzoyl precursors, the methyl 2,3,4-tri- O -benzoyl-pyranosides of ,- D -galactose, ,- D -galactose, ,- D -glucose, and methyl 2,3-di- O -benzoyl-,- D -galactopyranoside could be isolated in 85,96,% yield. In hydrolysis of methyl 2,3,4,6-tetra- O -benzoyl-,- D -glucopyranoside and methyl 2,3,4,6-tetra- O -benzoyl-,- D -galactopyranoside substrate inhibition were observed, which in part could be overcome by increasing the reaction volume. Methyl 2,3,4,6-tetra- O -benzoyl-,- D -glucopyranoside and methyl 2,3,4,6-tetra- O -benzoyl-,- D -mannopyranoside were poor substrates for Candida rugosa lipase and low degree of conversion towards products were obtained under all conditions. No acyl migration was detected in any of the products.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009) [source] 7,8-Diaminoperlargonic acid aminotransferase from Mycobacterium tuberculosis, a potential therapeutic targetFEBS JOURNAL, Issue 20 2006Characterization, inhibition studies Diaminopelargonic acid aminotransferase (DAPA AT), which is involved in biotin biosynthesis, catalyzes the transamination of 8-amino-7-oxononanoic acid (KAPA) using S -adenosyl- l -methionine (AdoMet) as amino donor. Mycobacterium tuberculosis DAPA AT, a potential therapeutic target, has been overproduced in Escherichia coli and purified to homogeneity using a single efficient step on a nickel-affinity column. The enzyme shows an electronic absorption spectrum typical of pyridoxal 5,-phosphate-dependent enzymes and behaves as a homotetramer in solution. The pH profile of the activity at saturation shows a single ionization group with a pKa of 8.0, which was attributed to the active-site lysine residue. The enzyme shows a Ping Pong Bi Bi kinetic mechanism with strong substrate inhibition with the following parameters: KmAdoMet = 0.78 ± 0.20 mm, KmKAPA = 3.8 ± 1.0 µm, kcat = 1.0 ± 0.2 min,1, KiKAPA = 14 ± 2 µm. Amiclenomycin and a new analogue, 4-(4c -aminocyclohexa-2,5-dien-1r -yl)propanol (referred to as compound 1), were shown to be suicide substrates of this enzyme, with the following inactivation parameters: Ki = 12 ± 2 µm, kinact = 0.35 ± 0.05 min,1, and Ki = 20 ± 2 µm, kinact = 0.56 ± 0.05 min,1, for amiclenomycin and compound 1, respectively. The inactivation was irreversible, and the partition ratios were 1.0 and 1.1 for amiclenomycin and compound 1, respectively, which make these inactivators particularly efficient. compound 1 (100 µg·mL,1) completely inhibited the growth of an E. coli C268bioA mutant strain transformed with a plasmid expressing the M. tuberculosis bioA gene, coding for DAPA AT. Reversal of the antibiotic effect was observed on the addition of biotin or DAPA. Thus, compound 1 specifically targets DAPA AT in vivo. [source] Characterization of cinnamyl alcohol dehydrogenase of Helicobacter pyloriFEBS JOURNAL, Issue 5 2005An aldehyde dismutating enzyme Cinnamyl alcohol dehydrogenases (CAD; 1.1.1.195) catalyse the reversible conversion of p -hydroxycinnamaldehydes to their corresponding alcohols, leading to the biosynthesis of lignin in plants. Outside of plants their role is less defined. The gene for cinnamyl alcohol dehydrogenase from Helicobacter pylori (HpCAD) was cloned in Escherichia coli and the recombinant enzyme characterized for substrate specificity. The enzyme is a monomer of 42.5 kDa found predominantly in the cytosol of the bacterium. It is specific for NADP(H) as cofactor and has a broad substrate specificity for alcohol and aldehyde substrates. Its substrate specificity is similar to the well-characterized plant enzymes. High substrate inhibition was observed and a mechanism of competitive inhibition proposed. The enzyme was found to be capable of catalysing the dismutation of benzaldehyde to benzyl alcohol and benzoic acid. This dismutation reaction has not been shown previously for this class of alcohol dehydrogenase and provides the bacterium with a means of reducing aldehyde concentration within the cell. [source] Deamidation of labile asparagine residues in the autoregulatory sequence of human phenylalanine hydroxylaseFEBS JOURNAL, Issue 5 2003Structural, functional implications Two dimensional electrophoresis has revealed a microheterogeneity in the recombinant human phenylalanine hydroxylase (hPAH) protomer, that is the result of spontaneous nonenzymatic deamidations of labile asparagine (Asn) residues [Solstad, T. and Flatmark, T. (2000) Eur. J. Biochem.267, 6302,6310]. Using of a computer algorithm, the relative deamidation rates of all Asn residues in hPAH have been predicted, and we here verify that Asn32, followed by a glycine residue, as well as Asn28 and Asn30 in a loop region of the N-terminal autoregulatory sequence (residues 19,33) of wt-hPAH, are among the susceptible residues. First, on MALDI-TOF mass spectrometry of the 24 h expressed enzyme, the E. coli 28-residue peptide, L15,K42 (containing three Asn residues), was recovered with four monoisotopic mass numbers (i.e., m/z of 3106.455, 3107.470, 3108.474 and 3109.476, of decreasing intensity) that differed by 1 Da. Secondly, by reverse-phase chromatography, isoaspartyl (isoAsp) was demonstrated in this 28-residue peptide by its methylation by protein- l -isoaspartic acid O -methyltransferase (PIMT; EC 2.1.1.77). Thirdly, on incubation at pH 7.0 and 37 °C of the phosphorylated form (at Ser16) of this 28-residue peptide, a time-dependent mobility shift from tR,,34 min to ,,31 min (i.e., to a more hydrophilic position) was observed on reverse-phase chromatography, and the recovery of the tR,,34 min species decreased with a biphasic time-course with t0.5 -values of 1.9 and 6.2 days. The fastest rate is compatible with the rate determined for the sequence-controlled deamidation of Asn32 (in a pentapeptide without 3D structural interference), i.e., a deamidation half-time of ,,1.5 days in 150 mm Tris/HCl, pH 7.0 at 37 °C. Asn32 is located in a cluster of three Asn residues (Asn28, Asn30 and Asn32) of a loop structure stabilized by a hydrogen-bond network. Deamidation of Asn32 introduces a negative charge and a partial ,-isomerization (isoAsp), which is predicted to result in a change in the backbone conformation of the loop structure and a repositioning of the autoregulatory sequence and thus affect its regulatory properties. The functional implications of this deamidation was further studied by site-directed mutagenesis, and the mutant form (Asn32,Asp) revealed a 1.7-fold increase in the catalytic efficiency, an increased affinity and positive cooperativity of L-Phe binding as well as substrate inhibition. [source] Mechanism of substrate inhibition in cellulose synergistic degradationFEBS JOURNAL, Issue 16 2001Priit Väljamäe ,A comprehensive experimental study of substrate inhibition in cellulose hydrolysis based on a well defined system is presented. The hydrolysis of bacterial cellulose by synergistically operating binary mixtures of cellobiohydrolase I from Trichoderma reesei and five different endoglucanases as well as their catalytic domains displays a characteristic substrate inhibition. This inhibition phenomenon is shown to require the two-domain structure of an intact cellobiohydrolase. The experimental data were in accordance with a mechanism where cellobiohydrolases previously bound to the cellulose by means of their cellulose binding domains are able to find chain ends by lateral diffusion. An increased substrate concentration at a fixed enzyme load will also increase the average diffusion distance/time needed for cellobiohydrolases to reach new chain ends created by endoglucanases, resulting in an apparent substrate inhibition of the synergistic action. The connection between the binding properties and the substrate inhibition is encouraging with respect to molecular engineering of the binding domain for optimal performance in biotechnological processes. [source] Batch kinetics and modelling of ethanolic fermentation of wheyINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 6 2005Salman Zafar Summary The fermentation of whey by Kluyveromyces marxianus strain MTCC 1288 was studied using varying lactose concentrations at constant temperature and pH. The increase in substrate concentration up to a certain limit was accompanied by an increase in ethanol formation, for example, at a substrate concentration of 10 g L,1, the production of ethanol was 0.618 g L,1 whereas at 50 g L,1 it was 3.98 g L,1. However, an increase in lactose concentration to 100 g L,1 led to a drastic decrease in product formation and substrate utilization. The maximum ethanol yield was obtained with an initial lactose concentration of 50 g L,1. A method of batch kinetics was utilized to formulate a mathematical model using substrate and product inhibition constants. The model successfully simulated the batch kinetics observed at S0 = 10 and 50 g L,1 but failed in case of S0 = 100 g L,1 because of strong substrate inhibition. [source] Substrate activation of butyrylcholinesterase and substrate inhibition of acetylcholinesterase by 3,3-dimethylbutyl- N - n -butylcarbamate and 2-trimethylsilyl-ethyl- N - n -butylcarbamateJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 1 2007Shyh-Ying Chiou Abstract Carbamates are used to treat Alzheimer's disease. These compounds inhibit acetylcholinesterase and butyrylcholinesterase. The goal of this work is to use the substrate analogs of butyrylcholinesterase, 3,3-dimethylbutyl- N - n -butylcarbamate (1) and 2-trimethylsilyl-ethyl- N - n -butylcarbamate (2) to probe the substrate activation mechanism of butyrylcholinesterase. Compounds 1 and 2 are characterized as the pseudo substrate inhibitors of acetylcholinesterase; however, compounds 1 and 2 are characterized as the essential activators of butyrylcholinesterase. Therefore, compounds 1 and 2 mimic the substrate in the acetylcholinesterase-catalyzed reactions, but the behavior of compounds 1 and 2 mimics the substrate activation in the butyrylcholinesterase-catalyzed reactions. © 2007 Wiley Periodicals, Inc. J Biochem Mol Toxicol 21:24,31, 2007; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20158 [source] EFFECT OF PHYSICAL FACTORS ON ACETIC ACID PRODUCTION IN BRETTANOMYCES STRAINSJOURNAL OF FOOD PROCESS ENGINEERING, Issue 2 2005C. CASTRO-MARTINEZ ABSTRACT Four species of Brettanomyces (intermedius, bruxellensis, custersianus, clausenii) were examined to ascertain their acetic acid production capacity. The results showed that B. bruxellensis was the strain with the best volumetric productivity ,and ,specific ,production ,rate ,(P = 0.065 gL,1 h,1; ,Vp = 0.43 gg,1h,1). The best kinetic parameters were reached (P = 0.133 gL,1 h,1; Yp/s = 0.23; Pmax = 11.64 gL,1) ,at ,an ,airflow ,of ,288 Lh,1,(0.6 vvm, ,OTR = 124 mgO2L,1,h,1), and substrate inhibition was not observed. The influence of temperature and agitation on acetic acid production by B. bruxellensis in a glucose medium was investigated at different levels, 26, 30, 34C and 250, 350, 450 rpm, respectively. Temperature and agitation were shown to be deci-sive factors (P < 0.05) in acetic acid production at 288 Lh,1(0.6 vvm, OTR = 124 mgO2L,1 h). The optimal conditions for a high volumetric productivity were 30C and 250 rpm, respectively. [source] Selective enhancement of the activity of C-terminally truncated, but not intact, acetylcholinesteraseJOURNAL OF NEUROCHEMISTRY, Issue 1 2008Martina Zimmermann Abstract Acetylcholinesterase (AChE) is one of the fastest enzymes approaching the catalytic limit of enzyme activity. The enzyme is involved in the terminal breakdown of the neurotransmitter acetylcholine, but non-enzymatic roles have also been described for the entire AChE molecule and its isolated C-terminal sequences. These non-cholinergic functions have been attributed to both the developmental and degenerative situation: the major form of AChE present in these conditions is monomeric. Moreover, AChE has been shown to lose its typical characteristic of substrate inhibition in both development and degeneration. This study characterizes a form of AChE truncated after amino acid 548 (T548-AChE), whose truncation site is homologue to that of a physiological form of T-AChE detected in fetal bovine serum that has lost its C-terminal moiety supposedly due to proteolytic cleavage. Peptide sequences covered by this C-terminal sequence have been shown to be crucially involved in both developmental and degenerative mechanisms in vitro. Numerous studies have addressed the structure,function relationship of the AChE C-terminus with T548-AChE representing one of the most frequently studied forms of truncated AChE. In this study, we provide new insight into the understanding of the functional characteristics that T548-AChE acquires in solution: T548-AChE is incubated with agents of varying net charge and molecular weight. Together with kinetic studies and an analysis of different molecular forms and aggregation states of T548-AChE, we show that the enzymatic activity of T548-AChE, an enzyme verging at its catalytic limit is, nonetheless, apparently enhanced by up to 800%. We demonstrate, first, how the activity of T548-AChE can be enhanced through agents that contain highly positive charged moieties. Moreover, the un-competitive mechanism of activity enhancement most likely involves the peripheral anionic site of AChE that is reflected in delayed substrate inhibition being observed for activity enhanced T548-AChE. The data provides evidence towards a mechanistic and functional link between the form of AChE unique to both development and degeneration and a C-terminal peptide of T-AChE acting under those conditions. [source] Substrate inhibition of Pediococcus acidilactici by glucose on a waste medium.LETTERS IN APPLIED MICROBIOLOGY, Issue 5 2003Simulations, experimental results Abstract Aims: The possibility of substrate inhibition by glucose on biomass and pediocin production was studied in cultures of Pediococcus acidilactici on a residual medium. Methods and Results: Calculation of the substrate inhibition coefficient in the context of microbial growth is generally laborious, and very prone to experimental error. However, a simulation combining logistic and Monod kinetics equations demonstrates that quantitative evidence for this type of inhibition, without the possibility of misinterpretation, can be obtained through the comparison of punctual preasymptotic productions as a function of substrate concentration. Significance and Impact of the Study: It was concluded that glucose had an inhibitory effect on growth, but not on bacteriocin production. [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] | |||||||||||||