PH-dependence and structure-activity relationships in the papain-catalysed hydrolysis of anilides.
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ABSTRACT:
SUBMITTER: Lowe G
PROVIDER: S-EPMC1177120 | biostudies-other | 1971 Aug
REPOSITORIES: biostudies-other
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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 taccalonolides are microtubule stabilizers isolated from plants of the genus Tacca that show potent in vivo antitumor activity and the ability to overcome multiple mechanisms of drug resistance. The most potent taccalonolide identified to date, AJ, is a semisynthetic product generated from the major plant metabolite taccalonolide A in a two-step reaction. The first step involves hydrolysis of taccalonolide A to generate taccalonolide B, and then this product is oxidized to generate an epoxide group at C-22-C-23. To generate sufficient taccalonolide AJ for in vivo antitumor efficacy studies, the hydrolysis conditions for the conversion of taccalonolide A to B were optimized. During purification of the hydrolysis products, we identified the new taccalonolide AO (1) along with taccalonolide I. When the same hydrolysis reaction was performed on a taccalonolide E-enriched fraction, four new taccalonolides, assigned as AK, AL, AM, and AN (2-5), were obtained in addition to the expected product taccalonolide N. Biological assays were performed on each of the purified taccalonolides, which allowed for increased refinement of the structure-activity relationship of this class of compounds.
| S-EPMC3761391 | biostudies-literature
Project description:BackgroundRecent interest in Populus as a source of renewable energy, combined with its numerous available pretreatment methods, has enabled further research on structural modification and hydrolysis. To improve the biodegradation efficiency of biomass, a better understanding of the relationship between its macroscopic structures and enzymatic process is important.ResultsThis study investigated mutant cell wall structures compared with wild type on a molecular level. Furthermore, a novel insight into the structural dynamics occurring on mutant biomass was assessed in situ and in real time by functional Atomic Force Microscopy (AFM) imaging. High-resolution AFM images confirmed that genetic pretreatment effectively inhibited the production of irregular lignin. The average roughness values of the wild type are 78, 60, and 30 nm which are much higher than that of the mutant cell wall, approximately 10 nm. It is shown that the action of endoglucanases would expose pure crystalline cellulose with more cracks for easier hydrolysis by cellobiohydrolase I (CBHI). Throughout the entire CBHI hydrolytic process, when the average roughness exceeded 3 nm, the hydrolysis mode consisted of a peeling action.ConclusionFunctional AFM imaging is helpful for biomass structural characterization. In addition, the visualization of the enzymatic hydrolysis process will be useful to explore the cell wall structure-activity relationships.
| S-EPMC7640438 | biostudies-literature
Project description:A T4 DNA ligase-catalysed oligonucleotide polymerisation process has been recently developed to enable the incorporation of multiple functional groups throughout a nucleic acid polymer. T4 DNA ligase requires ATP as a cofactor to catalyse phosphodiester bond formation during the polymerisation process. Herein, we describe the structure-activity relationship of ATP within the context of T4 DNA ligase-catalyzed oligonucleotide polymerisation. Using high-throughput sequencing, we study not only the influence of ATP modification on polymerisation efficiency, but also on the fidelity and sequence bias of the polymerisation process.
| S-EPMC5419680 | biostudies-literature
Project description:The presteady-state kinetic parameters of the interaction ofwild-type glucoamylase from Aspergillus niger (EC 3.2.1.3)with maltose were obtained and analysed in the pH range 3-7 withintervals of 0.25 pH units. In all cases the following three-step reaction scheme was found to apply. [Equation: see text]. The general result of the analysis of the presteady-state kinetics is that glucoamylase G1 is affected by the protonation states of three groups, with pK(a) values of 2.7, 4.5 and 5.7 in the free enzyme and of 2.7, 4.75 and 6.5 in the first enzyme-substrate complex. The protonation of the group in the enzyme-substrate complex with a pK(a) 6.5 hadno effect on k(2) (1640 s(-1)) or k(-2) (20+/-4 s(-1)), but resulted in a stronger enzyme-substrate interaction, due to a decrease of K(1) from 40 to 6.3 mM. In other words,when the substrate is bound, the pK(a) of the acidgroup changes to increase the fraction of reactive enzyme. Since this pK(a) parallels that of the Michaelis complex, known from the pH-dependence of k(cat), the group in question is most probably the catalytic acid Glu-179. Protonation of Glu-179 thus is of no importance in the second step, clearly indicating that this step represents a conformational change and not the actual hydrolysis step of the reaction. Protonation of the pK(a)=4.75 group leads to a small decrease in k(2) to 1090 s(-1), and also to minor changes in K(1). The group with pK(a)=2.7 leads toa major decrease of k(2), of which the limit may bezero, but shows no effect on K(1). Thus no differenceis seen between the pK(a) values of the free enzymeand of the first enzyme-substrate complex at low pH.
| S-EPMC1221186 | biostudies-other
Project description:1. The pH-dependence of the pepsin-catalysed hydrolysis of three peptide substrates was studied by using a method for the continuous monitoring of the formation of ninhydrin-positive products. 2. Two peptide acid substrates, N-acetyl-l-phenylalanyl-l-phenylalanine and N-acetyl-l-phenylalanyl-l-phenylalanyl-glycine, show apparent pK(a) values of 1.1 and 3.5 in the plots of k(0)/K(m) versus pH. By contrast a neutral substrate, N-acetyl-l-phenylalanyl-l-phenylalanine amide, shows apparent pK(a) values of 1.0 and 4.7. 3. Together with the data of the preceding paper (Knowles, Sharp & Greenwell, 1969), these results are taken to indicate that the rate of pepsin-catalysed hydrolysis is controlled by the ionization of two groups, which on the free enzyme have apparent pK(a) values of 1.0 and 4.7. It is apparent that the anions of peptide acid substrates are not perceptibly bound to the enzyme, resulting in apparent pK(a) values of 3.5 for the dependence of k(0)/K(m) for these materials.
| S-EPMC1184642 | biostudies-other
Project description:Current nano-SARs are often based on univariate assessments and fail to provide tiered views on ENM-induced bio-effects. Here we pioneered a multi-hierarchical nano-SAR assessment for a representative ENM, Fe2O3, by metabolomics and proteomics analyses. The established nano-SAR profile allows visualizing the contributions of 7 basic properties of Fe2O3 to its diverse bio-effects. For instance, while surface reactivity is responsible for Fe2O3-induced cell migration, the inflammatory effects of Fe2O3 are determined by aspect ratio (nanorods) or surface reactivity (nanoplates). These nano-SARs were examined in THP-1 cells and animal lungs, which allowed us to decipher the detailed mechanisms including NLRP3 inflammasome pathway and monocyte chemoattractant protein-1 dependent signaling. This study provides new insights for nano-SARs, which may facilitate the tailored design of ENMs to endow them with desired bio-effects.
2019-11-12 | PXD010614 | Pride
Project description:This study shows for the first time that boric acid catalyses the hydrolysis of peroxyacids, resulting in an approximately 12-fold increase in hydrolysis rate for both peracetic acid (PAA) and 3-chloroperbenzoic acid (MCPBA) when 0.1 M boric acid is present. The maximum rate of hydrolysis occurs at pH 9 and pH 8.4 for PAA and MCPBA respectively. In contrast, carbonate buffer does not enhance the rate of PAA hydrolysis. The reaction was followed by measuring the initial rate of hydrogen peroxide formation using a specific Ti(iv) complexation method. The study of the hydrolysis reaction requires the presence of 2 × 10-5 M each of ethylenediaminetetraacetic acid (EDTA) and ethylenediamine tetramethylene phosphonic acid (EDTMP) in all solutions in order to chelate metal ions across the full pH range (3 to 13) that would otherwise contribute to peroxyacid decomposition. Catalysis of peroxyacid hydrolysis is most likely effected by the triganol boric acid acting as a Lewis acid catalyst, associating with the peroxide leaving group in the transition state to reduce the leaving group basicity. The products of the reaction are the well characterised monoperoxoborate species and the parent carboxylic acid. Analysis of the pH and borate dependence data reveals that in addition to a catalytic pathway involving a single boric acid molecule, there is a significant pathway involving either (a) two boric acid molecules or (b) the polyborate species, B3O3(OH)4 -. Knowledge about catalytic mechanisms for the loss of peroxyacids through hydrolysis is important because they are widely used in reagents in a range of oxidation, bleaching and disinfection applications.
| S-EPMC10103745 | biostudies-literature
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
Project description:1. The pH-dependence of the kinetic constants of the carnitine acetyltransferase reaction has been investigated with the enzyme from pigeon breast muscle. 2. Michaelis constants for (-)-carnitine and acetyl-(-)-carnitine vary in a similar fashion in the pH range 6.0-9.0. A single ionizing group on the enzyme with an apparent pK7.2 is required in the basic form for binding of these substrates. 3. Binding of CoASH or acetyl-CoA raises the apparent pK of an ionizing group on the enzyme from 7.85 to 8.25. This group is probably not directly involved in forming the enzyme-substrate complex, but its microscopic environment is presumably altered. Another group in either the substrate or the free enzyme, with an apparent pK6.4, is needed in the basic form for optimum binding of CoA substrates. 4. This last group has been unequivocally identified as the 3'-phosphate of CoA, by showing that the K(m) of carnitine acetyltransferase for the substrate acetyl-3'-dephospho-CoA is independent of pH in the range 6.0-7.8. 5. V'(max.), the maximum velocity of the catalysed reaction between acetyl-CoA and (-)-carnitine, is constant between pH6.0 and 8.8. 6. The significance of these results in terms of a previously postulated reaction scheme for this enzyme is discussed.
| S-EPMC1270612 | biostudies-other