Kinetics of chymotrypsin- and papain-catalysed synthesis of [leucine]enkephalin and [methionine]enkephalin.
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ABSTRACT:
SUBMITTER: Kullmann W
PROVIDER: S-EPMC1153641 | biostudies-other | 1984 Jun
REPOSITORIES: biostudies-other
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Project description:1. The reaction of alpha-chymotrypsin with sodium periodate at pH5.0 has been investigated. The enzyme consumes 2 moles of periodate/mole, and there is a concomitant fall in enzymic activity (with respect to l-tyrosine ethyl ester) to 55% of that of the native enzyme. After 3hr. no further change is observed in periodate uptake or in catalytic activity. 2. The oxidized enzyme is a homogeneous preparation of partially active chymotrypsin. 3. In the oxidized enzyme, one of the two methionine residues in the molecule has been converted into its sulphoxide. It is this reaction only that is responsible for the loss of activity. 4. The rate constants for the enzyme-catalysed acylation and deacylation reactions are unaltered by oxidation of the enzyme, both for a non-specific substrate (p-nitrophenyl acetate), and for three specific substrates: N-acetyl-l-tryptophan ethyl ester, N-acetyl-l-tryptophanamide and N-acetyl-l-valine ethyl ester. 5. The K(m) values for the aromatic substrates with the oxidized enzyme are twice those with the native enzyme. No change in Michaelis constant is seen for the non-aromatic substrate N-acetyl-l-valine ethyl ester. 6. The evidence points to the oxidized methionine residue in the modified enzyme being situated in the locus of the active site at which aromatic (or bulky) side chains of the substrates are bound.
| S-EPMC1215192 | biostudies-other
Project description:Members of the serpin (serine protease inhibitor) superfamily have been identified in higher, multicellular eukaryotes, as well as in bacteria, although surveillance of available genome sequences indicates that bacterial serpin-encoding (ser) homologs are not widely distributed. In members of the genus Bifidobacterium this gene appears to be present in at least five, and perhaps up to nine, out of 30 species tested. Moreover, phylogenetic analysis using available bacterial and eukaryotic serpin sequences revealed that bifidobacteria specify serpins that form a separate clade. We characterized the ser210B locus of Bifidobacterium breve 210B, which consists of a number of genes, whose deduced protein products display significant similarity to proteins encoded by corresponding loci found in several other bifidobacteria. Northern hybridization, primer extension, micro array analysis, RT-PCR and Quantitative Real Time (qRT) - PCR analysis revealed that a 3.5 kb polycistronic mRNA, encompassing the ser210B operon with a single transcriptional start site, is strongly induced following treatment of B. breve 210B cultures with particular proteases. In contrast, transcription of the ser homolog of other bifidobacteria, such as Bifidobacterium longum subsp. infantis, Bifidobacterium dentium and B. longum subsp. longum, appears to be triggered by a different set of proteases
2010-03-09 | GSE20626 | GEO
Project description:Propylene carbonate can be used as a green solvent for the asymmetric synthesis of cyanohydrin trimethylsilyl ethers from aldehydes and trimethylsilyl cyanide catalysed by VO(salen)NCS, though reactions are slower in this solvent than the corresponding reactions carried out in dichloromethane. A mechanistic study has been undertaken, comparing the catalytic activity of VO(salen)NCS in propylene carbonate and dichloromethane. Reactions in both solvents obey overall second-order kinetics, the rate of reaction being dependent on the concentration of both the aldehyde and trimethylsilyl cyanide. The order with respect to VO(salen)NCS was determined and found to decrease from 1.2 in dichloromethane to 1.0 in propylene carbonate, indicating that in propylene carbonate, VO(salen)NCS is present only as a mononuclear species, whereas in dichloromethane dinuclear species are present which have previously been shown to be responsible for most of the catalytic activity. Evidence from ⁵¹V NMR spectroscopy suggested that propylene carbonate coordinates to VO(salen)NCS, blocking the free coordination site, thus inhibiting its Lewis acidity and accounting for the reduction in catalytic activity. This explanation was further supported by a Hammett analysis study, which indicated that Lewis base catalysis made a much greater contribution to the overall catalytic activity of VO(salen)NCS in propylene carbonate than in dichloromethane.
| S-EPMC2982084 | biostudies-literature
Project description:Members of the serpin (serine protease inhibitor) superfamily have been identified in higher, multicellular eukaryotes, as well as in bacteria, although surveillance of available genome sequences indicates that bacterial serpin-encoding (ser) homologs are not widely distributed. In members of the genus Bifidobacterium this gene appears to be present in at least five, and perhaps up to nine, out of 30 species tested. Moreover, phylogenetic analysis using available bacterial and eukaryotic serpin sequences revealed that bifidobacteria specify serpins that form a separate clade. We characterized the ser210B locus of Bifidobacterium breve 210B, which consists of a number of genes, whose deduced protein products display significant similarity to proteins encoded by corresponding loci found in several other bifidobacteria. Northern hybridization, primer extension, micro array analysis, RT-PCR and Quantitative Real Time (qRT) - PCR analysis revealed that a 3.5 kb polycistronic mRNA, encompassing the ser210B operon with a single transcriptional start site, is strongly induced following treatment of B. breve 210B cultures with particular proteases. In contrast, transcription of the ser homolog of other bifidobacteria, such as Bifidobacterium longum subsp. infantis, Bifidobacterium dentium and B. longum subsp. longum, appears to be triggered by a different set of proteases Transcriptional response to protease treatments (kallikrein, papain and chymotrypsin) of Bifidobacterium breve 210B
2010-05-06 | E-GEOD-20626 | biostudies-arrayexpress
Project description:Reactions involving hydrophobic reactants in water can be much accelerated in organic solvent-free solutions containing amphiphiles at neutral pH and room temperature. Previously, we demonstrated that organosilica colloidal particles could be conveniently synthesized by a versatile salt-catalysis method in solutions modified with various amphiphilic molecules. The method precludes the use of any solvent, any added form of energy (thermal or mechanical), and any strong (or hazardous) acids/bases. Herein, the kinetic properties of the reaction were systematically investigated for fluoride-catalysed synthesis of colloidal organosilica from a thiol-functionalized organosilane precursor, (3-mercaptopropyl)trimethoxysilane. Continuous, real-time ATR-FTIR measurements allowed probing the time evolution of organosilica condensation in different reaction systems, containing one of the following: non-ionic surfactants (Tween 20, Tween 40, Tween 60, Tween 80, Triton X-100), anionic surfactant (sodium dodecyl sulphate; SDS), cationic surfactant (cetyltrimethylammonium bromide; CTAB), and amphiphilic polymers (polyvinyl alcohol and polyvinylpyrrolidone). Overall, while some amphiphile-specific properties were revealed, fluoride-catalysed synthesis was ultrafast with a universal two-phase kinetic scheme (e.g. transition within 5-10 min) for all amphiphiles studied.
| S-EPMC9070784 | biostudies-literature
Project description:1. The maximum rate of production of p-nitrophenol (V(max.)) for both papain- and ficin-catalysed hydrolyses of p-nitrophenyl hippurate is independent of methanol concentration up to 2m for papain and 1.5m for ficin. 2. The observed catalytic constant (k(0)) for the production of hippuric acid for both papain- and ficin-catalysed hydrolyses of methyl hippurate decreases with increasing methanol concentration, 1/k(0) being linearly dependent on the methanol concentration. The k(MeOH)/k(H2O) ratio is determined. 3. These results provide strong evidence against general base catalysis for the rate-determining step in the deacylation of hippuryl-papain and hippuryl-ficin and probably for other specific acyl-papains and acyl-ficins. 4. The rate-determining step for the deacylation of the non-specific trans-cinnamoyl-papain appears to be different from that for the specific hippuryl-papain, and is probably subject to general base catalysis. It is possible, however, to accommodate all these observations in a single four-step reaction pathway. 5. Propan-2-ol did not influence the rate of production of hippuric acid for the papain-catalysed hydrolysis of methyl hippurate. A similar result has previously been reported for the ficin-catalysed hydrolysis of methyl hippurate. Ethanol and of course methanol (see 2) decrease the rate of production of hippuric acid for both papain- and ficin-catalysed hydrolyses of methyl hippurate. It is suggested that the secondary alcohol is incapable for structural reasons of approaching the bond to be hydrolysed.
| S-EPMC1270121 | biostudies-other
Project description:ObjectiveThe aim of this work was to investigate the immunological effect of MENK by analyzing the protein spectrum and bioinformatics of macrophage RAW264.7, and to explore the relationship between macrophage and ferroptosis.ResultWe employed proteomic analysis to identify differentially expressed proteins (DEPs) between macrophages and macrophages intervened by MENK. A total of 208 DEPs were identified. Among these, 96 proteins had upregulated expression and 112 proteins had downregulated expression. Proteomic analysis revealed a significant enrichment of DEPs associated with iron metabolism. The identification of hub genes was conducted using KEGG pathway diagrams and protein-protein interaction (PPI) analysis. The hub genes identified in this study include HMOX1 and Ferritin (FTH and FTL). A correlation was established between HMOX1, FTH, and FTL in the GO and KEGG databases. The results of PCR, WB and immunofluorescence showed that MENK downregulated the level of HMOX1 and FTH.ConclusionMENK had the potential to become an adjuvant chemotherapy drug by regulating iron metabolism in macrophages, reducing levels of HMOX1 and ferritin. We proposed an innovative research direction on the therapeutic potential of MENK, focusing on the relationship between ferroptosis and macrophage activity.
| S-EPMC10799539 | biostudies-literature
Project description:1. The Michaelis-Menten parameters for the papain-catalysed hydrolysis of a number of alkyl, aryl and alkyl-thiol esters of hippuric acid have been determined. 2. For all the aryl esters and most of the alkyl esters studied, the catalytic constant, k(0), is 2-3sec.(-1) and most probably represents deacylation of the common intermediate, hippuryl-papain. 3. Two alkyl esters and hippurylamide, however, have catalytic rate constants, k(0), less than 2-3sec.(-1). It is possible to interpret all the available kinetic data in terms of a three-step mechanism in which an enzyme-substrate complex is first formed, followed by acylation of the enzyme through an essential thiol group, followed by deacylation of the acyl-enzyme. 4. The logarithm of the ratio of the Michaelis-Menten parameters, which reflect the acylation rate constant, for four aryl esters of hippuric acid studied give a linear Hammett plot against the substituent constant, sigma. Arguments are presented that indicate acid as well as nucleophilic catalysis in the acylation process and that the most likely proton donor is an imidazolium ion. 5. It is suggested that this imidazolium ion is part of the same histidine residue that has been tentatively implicated in the deacylation process (Lowe & Williams, 1965b). 6. A new mechanism is proposed for the papain-catalysed hydrolysis of N-acyl-alpha-amino acid derivatives.
| S-EPMC1206922 | biostudies-other
Project description:The specificity of the proteolytic enzyme, papain, for the peptide bond of the substrate adjacent to that about to be cleaved and for the acyl residue of some N-acylglycine derivatives is manifest almost exclusively in the formation of the acyl-enzyme from the enzyme-substrate complex. Models for the enzyme-substrate complex and acyl-enzyme intermediate are suggested that account for these observations. In particular it is suggested that the peptide bond of the substrate adjacent to that about to be cleaved, is bound in the cleft of the enzyme between the NH group of glycine-66 and the backbone C=O group of aspartic acid-158, and provides a sensitive amplification mechanism through which the specificity of the enzyme for hydrophobic amino acids such as l-phenylalanine is relayed. It is also suggested that the distortion in the enzyme-substrate complex and the binding of the peptide bond adjacent to that about to be cleaved are also linked and behave co-operatively, the distortion of the protein facilitating binding and the stronger binding facilitating distortion. The results imply that between the enzyme-substrate complex and the acyl-enzyme a relaxation of the protein conformation must occur.
| S-EPMC1177119 | biostudies-other
Project description:The chemical synthesis of N-alpha-benzyloxycarbonyl-L-lysine p-nitroanilide (Z-Lys-pNA) is described in detail. The pH-dependence of the catalytic parameters kcat,' Km and kcat./Km for the papain-catalysed hydrolysis of Z-Lys-pNA are determined. kcat. and Km are pH-independent between pH 5 and pH 7.42, but the pH-dependence of kcat./Km is bell-shaped, decreasing at high and low pH values with pKa values of 7.97 and 4.40 respectively. The catalytic parameters and their pH-dependence are shown to be similar to those reported for other anilide substrates and it is concluded that the Km value of 0.01 mM previously reported [Angelides & Fink (1979) Biochemistry 18, 2355-2369] is incorrect. The possibility of accumulating a tetrahedral intermediate during the papain-catalysed hydrolysis of Z-Lys-pNA is discussed.
| S-EPMC1144749 | biostudies-other