Stoichiometry of reactions of alpha 2-macroglobulin with trypsin and chymotrypsin.
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ABSTRACT:
SUBMITTER: Bjork I
PROVIDER: S-EPMC1153210 | biostudies-other | 1984 Jan
REPOSITORIES: biostudies-other
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Project description:Previous interpretations of the mechanism of trypsin- and chymotrypsin-catalysed reactions in terms of two intermediates, the Michaelis complex and an acyl-enzyme, were based on steady-state studies and on the observation of individual steps under sub-optimum conditions. In the present paper new methods for the rapid analysis of chemical events and for the spectrophotometric detection of individual steps are applied to these two enzymes. These methods can be used to study reactions with specific amino acid ester substrates. It can be shown that there are at least three distinct steps between the Michaelis complex and the release of ethanol; the latter is likely to correspond to acyl-enzyme formation. The relative rates of these three steps are measured by rapid-flow techniques from observations of the displacement of chromophoric inhibitors and reactions with specific substrates containing chromophores, as well as from ethanol analyses during a single turnover of the enzyme reactions. It is concluded that the reactions of trypsin and chymotrypsin with their specific substrates involve the formation of a specially reactive conformation of the enzyme-substrate complex and that the rate constants involved in this rearrangement are at least as important for the overall reaction as those of the subsequent formation and decomposition of the acyl-enzyme.
| S-EPMC1270122 | biostudies-other
Project description:Mesotrypsin is an unusual human trypsin isoform with inhibitor resistance and the ability to degrade trypsin inhibitors. Degradation of the protective serine protease inhibitor Kazal type 1 (SPINK1) by mesotrypsin in the pancreas may contribute to the pathogenesis of pancreatitis. Here we tested the hypothesis that the regulatory digestive protease chymotrypsin C (CTRC) mitigates the harmful effects of mesotrypsin by cleaving the autolysis loop. As human trypsins are post-translationally sulfated in the autolysis loop, we also assessed the effect of this modification. We found that mesotrypsin cleaved in the autolysis loop by CTRC exhibited catalytic impairment on short peptides due to a 10-fold increase in Km , it digested β-casein poorly and bound soybean trypsin inhibitor with 10-fold decreased affinity. Importantly, CTRC-cleaved mesotrypsin degraded SPINK1 with markedly reduced efficiency. Sulfation increased mesotrypsin activity but accelerated CTRC-mediated cleavage of the autolysis loop and did not protect against the detrimental effect of CTRC cleavage. The observations indicate that CTRC-mediated cleavage of the autolysis loop in mesotrypsin decreases protease activity and thereby protects the pancreas against unwanted SPINK1 degradation. The findings expand the role of CTRC as a key defense mechanism against pancreatitis through regulation of intrapancreatic trypsin activity.
| S-EPMC7076226 | biostudies-literature
Project description:Brief digestion of ox neurofilaments with trypsin liberates fragments that are soluble and have molecular weights ranging from 164 000 to 97 000. Peptide fingerprinting indicates that these regions, termed the tryptic head-regions, arise from the 205 000- and 158 000-mol.wt. components of the triplet. The remains of the parent polypeptides sediment with normal filaments and have been termed tail-regions. Digestion of neurofilaments with chymotrypsin also liberates soluble fragments (chymotryptic head-regions) but these have mol.wts. 171 000 and 119 000, though they too originate from the higher-molecular-weight triplet polypeptides. Tryptic and chymotryptic head-regions have extensive homology, and a low (less than or equal to 20%) helix content. Electron microscopy shows that chymotryptic digestion rapidly reduces the length of filaments, probably because this enzyme preferentially attacks the 72 000-mol.wt. polypeptide. In contrast, brief digestion with trypsin does not reduce filament length even though more than 90% of the two higher-molecular-weight components have been cleaved. These results indicate that the backbone of native filaments is formed from the 72 000-mol.wt. polypeptide together with the tail-regions from the 205 000- and 158 000-mol.wt. polypeptides. The corresponding head-regions of these components, which can represent nearly 75% of each molecule, are not necessary for preserving the backbone of native neurofilaments and are therefore good candidates for being the side arms that connect these filaments in nerve cells.
| S-EPMC1152391 | biostudies-other
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:BackgroundBlood flukes (Schistosoma spp.) are parasites that can survive for years or decades in the vasculature of permissive mammalian hosts, including humans. Proteolytic enzymes (proteases) are crucial for successful parasitism, including aspects of invasion, maturation and reproduction. Most attention has focused on the 'cercarial elastase' serine proteases that facilitate skin invasion by infective schistosome larvae, and the cysteine and aspartic proteases that worms use to digest the blood meal. Apart from the cercarial elastases, information regarding other S. mansoni serine proteases (SmSPs) is limited. To address this, we investigated SmSPs using genomic, transcriptomic, phylogenetic and functional proteomic approaches.Methodology/principal findingsGenes encoding five distinct SmSPs, termed SmSP1 - SmSP5, some of which comprise disparate protein domains, were retrieved from the S. mansoni genome database and annotated. Reverse transcription quantitative PCR (RT- qPCR) in various schistosome developmental stages indicated complex expression patterns for SmSPs, including their constituent protein domains. SmSP2 stood apart as being massively expressed in schistosomula and adult stages. Phylogenetic analysis segregated SmSPs into diverse clusters of family S1 proteases. SmSP1 to SmSP4 are trypsin-like proteases, whereas SmSP5 is chymotrypsin-like. In agreement, trypsin-like activities were shown to predominate in eggs, schistosomula and adults using peptidyl fluorogenic substrates. SmSP5 is particularly novel in the phylogenetics of family S1 schistosome proteases, as it is part of a cluster of sequences that fill a gap between the highly divergent cercarial elastases and other family S1 proteases.Conclusions/significanceOur series of post-genomics analyses clarifies the complexity of schistosome family S1 serine proteases and highlights their interrelationships, including the cercarial elastases and, not least, the identification of a 'missing-link' protease cluster, represented by SmSP5. A framework is now in place to guide the characterization of individual proteases, their stage-specific expression and their contributions to parasitism, in particular, their possible modulation of host physiology.
| S-EPMC3967958 | biostudies-literature
Project description:A ternary complex of the black-eyed pea trypsin and chymotrypsin inhibitor (BTCI) with trypsin and chymotrypsin was crystallized by the sitting-drop vapour-diffusion method with 0.1 M HEPES pH 7.5, 10%(w/v) polyethylene glycol 6000 and 5%(v/v) 2-methyl-2,4-pentanediol as precipitant. BTCI is a small protein with 83 amino-acid residues isolated from Vigna unguiculata seeds and is able to inhibit trypsin and chymotrypsin simultaneously by forming a stable ternary complex. X-ray data were collected from a single crystal of the trypsin-BTCI-chymotrypsin ternary complex to 2.7 A resolution under cryogenic conditions. The structure of the ternary complex was solved by molecular replacement using the crystal structures of the BTCI-trypsin binary complex (PDB code 2g81) and chymotrypsin (PDB code 4cha) as search models.
| S-EPMC2344091 | biostudies-literature
Project description:Abstract The food enzyme is a serine protease complex, containing trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1), obtained from porcine pancreas by Neova Technologies Inc. The serine protease complex is intended to be used for hydrolysis of whey proteins employed as ingredients of infant formulae and follow?on formulae. Based on maximum use levels and the maximum permitted protein content in infant formulae, dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be 18 mg TOS/kg body weight (bw) per day for infants. In the toxicological evaluation, clinical studies with pharmaceutical preparations containing pancreatic enzymes were considered. Hypersensitivity to the pharmaceuticals was identified as the major side effect. However, allergic reactions to porcine pancreatic enzymes in hydrolysed foods have not been reported. The Panel considered that a risk of allergic sensitisation to this food enzyme after consumption of products prepared by hydrolysis of milk cannot be excluded in infants, but considers the likelihood to be low. Based on the origin of the food enzyme from edible parts of animals, the data provided by the applicant, supported by the evaluation of clinical studies with pharmaceutical preparations based on pancreatic enzymes, the Panel concluded that the porcine pancreatic enzymes do not give rise to safety concern under the intended conditions of use.
| S-EPMC7787161 | biostudies-literature
Project description:Trypsin and chymotrypsin are both serine proteases with high sequence and structural similarities, but with different substrate specificity. Previous experiments have demonstrated the critical role of the two loops outside the binding pocket in controlling the specificity of the two enzymes. To understand the mechanism of such a control of specificity by distant loops, we have used the Gaussian network model to study the dynamic properties of trypsin and chymotrypsin and the roles played by the two loops. A clustering method was introduced to analyze the correlated motions of residues. We have found that trypsin and chymotrypsin have distinct dynamic signatures in the two loop regions, which are in turn highly correlated with motions of certain residues in the binding pockets. Interestingly, replacing the two loops of trypsin with those of chymotrypsin changes the motion style of trypsin to chymotrypsin-like, whereas the same experimental replacement was shown necessary to make trypsin have chymotrypsin's enzyme specificity and activity. These results suggest that the cooperative motions of the two loops and the substrate-binding sites contribute to the activity and substrate specificity of trypsin and chymotrypsin.
| S-EPMC1366603 | biostudies-literature
Project description:Cyanopeptolins (CPs) are one of the most frequently occurring cyanobacterial peptides, many of which are inhibitors of serine proteases. Some CP variants are also acutely toxic to aquatic organisms, especially small crustaceans. In this study, thirteen CPs, including twelve new variants, were detected in the cyanobacterium Nostoc edaphicum CCNP1411 isolated from the Gulf of Gdańsk (southern Baltic Sea). Structural elucidation was performed by tandem mass spectrometry with verification by NMR for CP962 and CP985. Trypsin and chymotrypsin inhibition assays confirmed the significance of the residue adjacent to 3-amino-6-hydroxy-2-piperidone (Ahp) for the activity of the peptides. Arginine-containing CPs (CPs-Arg²) inhibited trypsin at low IC50 values (0.24⁻0.26 µM) and showed mild activity against chymotrypsin (IC50 3.1⁻3.8 µM), while tyrosine-containing CPs (CPs-Tyr²) were selectively and potently active against chymotrypsin (IC50 0.26 µM). No degradation of the peptides was observed during the enzyme assays. Neither of the CPs were active against thrombin, elastase or protein phosphatase 1. Two CPs (CP962 and CP985) had no cytotoxic effects on MCF-7 breast cancer cells. Strong and selective activity of the new cyanopeptolin variants makes them potential candidates for the development of drugs against metabolic disorders and other diseases.
| S-EPMC6070996 | biostudies-literature
Project description:The food enzyme is a serine protease complex containing trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) obtained from porcine pancreas by Paninkret Chem.-Pharm. Werk GmbH. The food enzyme is currently only used in protein processing to hydrolyse milk proteins. Milk protein hydrolysates and peptides are mainly used in formulae intended to have reduced allergenicity. Based on the recommended use level and the high consumption of formulae in very young babies, dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be 180 mg TOS/kg body weight (bw) per day for infants and toddlers. Toxicological evaluation was based on the available clinical studies with pancreatic enzymes. Hypersensitivity to the product was identified as the major side effect. However, the intact enzymes are inactivated during preparation of food products; therefore, the Panel considered that the likelihood of adverse effects of the intact enzyme to occur is low. The Panel considered that a risk of allergic sensitisation to these protein hydrolysates after consumption cannot be excluded, but the likelihood of occurrence was considered to be low. Based on the origin of the food enzyme from edible parts of animals, the data provided and the evaluation of clinical studies with pancreatic enzymes and the estimated dietary exposure, the Panel concluded that the food enzyme does not give rise to safety concerns when used in the production of infant formulae based on milk protein hydrolysates.
| S-EPMC8207344 | biostudies-literature