The pathway of pepsin-catalysed transpeptidation. Evidence for the reactive species being the anion of the acceptor molecule.
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ABSTRACT:
SUBMITTER: Kitson TM
PROVIDER: S-EPMC1176769 | biostudies-other | 1971 Apr
REPOSITORIES: biostudies-other
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Project description:The action of pig pepsin on a variety of small peptides including Leu-Trp-Met-Arg, Leu-Trp-Met, Leu-Leu-NH2, benzyloxycarbonyl-Phe-Leu and Gly-Leu-Tyr was studied. Leu-Leu-Leu was found to be the major product from the substrates Leu-Trp-Met-Arg and Leu-Trp-Met, indicating that the predominant reaction at pH 3.4 was a transpeptidation of the acyl-transfer type. Leu-Leu-Leu was also formed in high yield by amino transfer from benzyloxycarbonyl-Phe-Leu. Like the amino-transfer reactions the acyl transfer proceeded via a covalent intermediate, since [14C]leucine was not incorporated into transpeptidation products and did not exchange with enzyme-bound leucine in the presence of acceptors. With Leu-Trp-Met both acyl and amino transpeptidation products, namely Leu-Leu, Leu-Leu-Leu, Met-Met and Met-Met-Met, were formed in addition to methionine and leucine. With Leu-Trp-Met-Arg (1 mM) the pH optimum for the rates of hydrolysis and acyl transfer is about pH 3.4. At this pH the rate of acyl transfer exceeds that of hydrolysis; at pH 2, however, hydrolysis was faster than transfer. A comparison of the effect of the length of substrates and products on the reaction rates allows the conclusion that the binding site can extend over eight to nine amino acid residues. Although the experiments provide no conclusive evidence for or against the involvement of amino and/or acyl intermediates in the hydrolysis of long peptides and proteins, the high yield of transpeptidation reactions of both types observed with some substrates suggests a major role for the intermediates in pepsin-catalysed reactions. The results also show that when pig pepsin is used for the digestion of proteins for sequence work, the likelihood of the formation of transpeptidation products is considerable. In this way peptides not present in the original sequence could easily form in a reasonably good yield.
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Project description:The glycylglycine endopeptidase in lysostaphin has been found capable of catalysing both hydrolysis and transpeptidation reactions when acting on glycyl peptides. The ability of the enzyme to utilize dansyldiglycine (5-dimethylaminoaphthalene-1-sulphonylglycylglycine) as an acceptor molecule in transpeptidation reactions, although it is incapable of hydrolysing the peptide bond in this compound, indicates the enzyme must be capable of forming the equivalent of an imino-enzyme intermediate during the catalytic process.
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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.
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Project description:The intrinsic apoptosis pathway is conserved from worms to humans and plays a critical role in the normal development and homeostatic control of adult tissues. As a result, numerous diseases from cancer to neurodegeneration are associated with either too little or too much apoptosis.B cell lymphoma-2 (BCL-2) family members regulate cell death, primarily via their effects on mitochondria. In stressed cells, proapoptotic BCL-2 family members promote mitochondrial outer membrane permeabilization (MOMP) and cytochrome c (cyt c) release into the cytoplasm, where it stimulates formation of the "apoptosome." This large, multimeric complex is composed of the adapter protein, apoptotic protease-activating factor-1, and the cysteine protease, caspase-9. Recent studies suggest that proteins involved in the processes leading up to (and including) formation of the apoptosome are subject to various forms of post-translational modification, including proteolysis, phosphorylation, and in some cases, direct oxidative modification.Despite intense investigation of the intrinsic pathway, significant questions remain regarding how cyt c is released from mitochondria, how the apoptosome is formed and regulated, and how caspase-9 is activated within the complex.Further studies on the biochemistry of MOMP and apoptosome formation are needed to understand the mechanisms that underpin these critical processes, and novel animal models will be necessary in the future to ascertain the importance of the many posttranslational modifications reported for BCL-2 family members and components of the apoptosome.
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Project description:Background: Acquired resistance to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) such as erlotinib is a major challenge to achieve an overall clinical benefit of the targeted therapy. Recently, aldehyde dehydrogenase 1 (ALDH1) induction has been found to render lung adenocarcinomas resistant to EGFR-TKIs, and targeting ALDH1A1 becomes a novel strategy to overcome resistance. However, the molecular mechanism underlying such effect remains poorly understood. Methods: Comprehensive assays were performed in a panel of lung adenocarcinoma cell lines and xenografts that acquired resistance to erlotinib. Cancer phenotype was evaluated by cell viability, apoptosis, migration, and epithelial-mesenchymal transition analysis in vitro, tumorsphere formation analysis ex vivo, and tumor growth and dissemination analysis in vivo. Reactive oxygen species (ROS) and reactive carbonyl species (RCS) were detected based on fluorescent oxidation indicator and liquid chromatography coupled to mass spectrometry, respectively. Protein target was suppressed by RNA interference and pharmacological inhibition or ecto-overexpressed by lentivirus-based cloning. Gene promoter activity was measured by dual-luciferase reporting assay. Results: Knockdown or pharmacological inhibition of ALDH1A1 overcame erlotinib resistance in vitro and in vivo. ALDH1A1 overexpression was sufficient to induce erlotinib resistance. Metabolomic analysis demonstrated lower ROS-RCS levels in ALDH1A1-addicted, erlotinib-resistant cells; in line with this, key enzymes for metabolizing ROS and RCS, SOD2 and GPX4, respectively, were upregulated in these cells. Knockdown of SOD2 or GPX4 re-sensitized the resistant cells to erlotinib and the effect was abrogated by ROS-RCS scavenging and mimicked by ROS-RCS induction. The ALDH1A1 overexpressed cells, though resisted erlotinib, were more sensitive to SOD2 or GPX4 knockdown. The ALDH1A1 effect on erlotinib resistance was abrogated by ROS-RCS induction and mimicked by ROS-RCS scavenging. Detection of GPX4 and SOD2 expression and analysis of promoter activities of GPX4 and SOD2 under the condition of suppression or overexpression of ALDH1A1 demonstrated that the RCS-ROS-metabolic pathway was controlled by the ALDH1A1-GPX4-SOD2 axis. The ROS-RCS metabolic dependence mechanism in ALDH1A1-induced resistance was confirmed in vivo. Analysis of public databases showed that in patients undergoing chemotherapy, those with high co-expression of ALDH1A1, GPX4, and SOD2 had a lower probability of survival. Conclusions: ALDH1A1 confers erlotinib resistance by facilitating the ROS-RCS metabolic pathway. ALDH1A1-induced upregulation of SOD2 and GPX4, as well as ALDH1A1 itself, mitigated erlotinib-induced oxidative and carbonyl stress, and imparted the TKI resistance. The elucidation of previously unrecognized metabolic mechanism underlying erlotinib resistance provides new insight into the biology of molecular targeted therapies and help to design improved pharmacological strategies to overcome the drug resistance.
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