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.
Project description:1. p-Nitrophenyl N(2)-acetyl-N(1)-benzylcarbazate (NPABC) was synthesized and shown to acylate alpha-chymotrypsin stoicheiometrically; reaction at 25 degrees occurs almost instantaneously at pH7.04 and within 2min. at pH5.04 and there is no observable turnover during 10min. 2. The absolute molarity of solutions of alpha-chymotrypsin can be determined by spectrophotometric measurement of the p-nitrophenol liberated during the acylation step; the results obtained at pH5.04 and pH7.04 agree with one another and with those determined by the method of Erlanger & Edel (1964). 3. Trypsin reacts stoicheiometrically, but more slowly than alpha-chymotrypsin, with NPABC, and it, like chymotrypsin, can be spectrophotometrically titrated at pH7.04. At pH5.04, however, reaction between trypsin and NPABC is sufficiently slow for the reagent to be nearly specific for alpha-chymotrypsin. Specificity for one or other enzyme can be ensured by using soya-bean trypsin inhibitor or the chymotrypsin inhibitor l-1-chloro-3-toluene-p-sulphonamido-4-phenylbutan-2-one. Bovine thrombin does not react with NPABC. 4. Evidence is presented that indicates that acylation of alpha-chymotrypsin and trypsin by NPABC occurs at the active centres of the enzymes. 5. Evidence was obtained that indicates that one or more tryptophan residues move into a more hydrophobic environment when alpha-chymotrypsin and trypsin are acylated by NPABC.
Project description:Sweet potato proteins have been shown to possess antioxidant and antidiabetic properties in vivo. The ability of a protein to exhibit systemic effects is somewhat unusual as proteins are typically susceptible to digestive enzymes. This study was undertaken to better understand how digestive enzymes affect sweet potato proteins. Two fractions of industrially processed sweet potato peel, containing 6.8% and 8.5% protein and 80.5% and 83.3% carbohydrate, were used as a source of protein. Sweet potato proteins were incubated with pepsin, trypsin, and chymotrypsin and protein breakdown was visualized with SDS-PAGE. After pepsin digestion, samples were assayed for amylase inhibitory activity. Sporamin, the major storage protein in sweet potatoes, which functions as a trypsin inhibitor as well, exhibited resistance to pepsin, trypsin, and chymotrypsin. Sporamin from blanched peel of orange sweet potatoes was less resistant to pepsin digestion than sporamin from outer peel and from extract of the white-skinned Caiapo sweet potato. Trypsin inhibitory activity remained after simulated gastric digestion, with the Caiapo potato protein and peel samples exhibiting higher inhibitory activity compared to the blanched peel sample. Amylase and chymotrypsin inhibitory activity was not present in any of the samples after digestion.
Project description:Human noroviruses (HuNoVs) are the leading cause of acute gastroenteritis worldwide. Histo-Blood Groups Antigens (HBGAs) have been described as attachment factors, promoting HuNoV infection. However, their role has not yet been elucidated. This study aims to evaluate the ability of HBGAs to protect HuNoVs against various factors naturally found in the human digestive system. The effects of acid pH and proteolytic enzymes (pepsin, trypsin, and chymotrypsin) on GII.4 virus-like particles (VLPs) and GII.4 HuNoVs were studied, both during interactions and non-interaction with HBGAs. The results showed that GII.4 VLPs and GII.4 HuNoVs behaved differently following the treatments. GII.4 VLPs were disrupted at a pH of less than 2.0 and in the presence of proteolytic enzymes (1,500 units/mL pepsin, 100 mg/mL trypsin, and 100 mg/mL chymotrypsin). VLPs were also partially damaged by lower concentrations of trypsin and chymotrypsin (0.1 mg/mL). Conversely, the capsids of GII.4 HuNoVs were not compromised by such treatments, since their genomes were not accessible to RNase. HBGAs were found to offer GII.4 VLPs no protection against an acid pH or proteolytic enzymes.
Project description:The autolysis of trypsin and alpha-chymotrypsin is accelerated in the presence of colloidal silica and glass surfaces. It is proposed that adsorption of the enzymes (favoured by electrostatic factors) results in a conformational change that renders the adsorbed enzyme more susceptible to proteolytic attack. Although the adsorbed enzymes are more susceptible to proteolysis, their activity towards low-molecular-weight substrates is not affected, indicating a relatively minor conformational change on adsorption. The rates of autolysis in solution (i.e. in ;inert' vessels) are second-order for both trypsin and alpha -chymotrypsin, with rate constants of 13.0mol(-1).dm(3).s(-1) for trypsin (in 50mm-NaCl at pH8.0 at 25 degrees C) and 10.2mol(-1).dm(3).s(-1) for alpha-chymotrypsin (in 0.1m-glycine at pH9.2 at 30 degrees C). In glass vessels or in the presence of small areas of silica surface (as colloidal silica particles), the autolysis of both trypsin and alpha-chymotrypsin can show first-order kinetics. Under these conditions, saturation of the surface occurs and the fast surface proteolytic reaction controls the overall kinetic order. However, when greater areas of silica surface are present, saturation of the surface does not occur, and, since for a considerable portion of the adsorption isotherm the amount adsorbed is approximately proportional to the concentration in solution, second-order kinetics are again observed. A number of negatively charged macromolecules have been shown similarly to increase the rate of autolysis of trypsin: thus this effect, observed initially with glass and silica surfaces, is of more general occurrence when these enzymes adsorb on or interact with negatively charged surfaces and macromolecules. These observations explain the confusion in the literature with regard to the kinetics of autolysis of alpha-chymotrypsin, where first-order, second-order and intermediate kinetics have been reported. A further effect of glass surfaces and negatively charged macromolecules is to shift the pH-activity curve of trypsin to higher pH values, as a consequence of the effective decrease in pH in the ;microenvironment' of the enzyme associated with the negatively charged surface or macromolecule.
Project description:The field of proteomics almost uniformly relies on peptide cation analysis, leading to an underrepresentation of acidic portions of proteomes, including relevant acidic posttranslational modifications. Despite the many benefits negative mode proteomics can offer, peptide anion analysis remains in its infancy due mainly to challenges with high-pH reversed-phase separations and a lack of robust fragmentation methods suitable for peptide anion characterization. Here, we report the first implementation of activated ion negative electron transfer dissociation (AI-NETD) on the chromatographic timescale, generating 7,601 unique peptide identifications from Saccharomyces cerevisiae in single-shot nLC-MS/MS analyses of tryptic peptides-a greater than 5-fold increase over previous results with NETD alone. These improvements translate to identification of 1,106 proteins, making this work the first negative mode study to identify more than 1,000 proteins in any system. We then compare the performance of AI-NETD for analysis of peptides generated by five proteases (trypsin, LysC, GluC, chymotrypsin, and AspN) for negative mode analyses, identifying as many as 5,356 peptides (1,045 proteins) with LysC and 4,213 peptides (857 proteins) with GluC in yeast-characterizing 1,359 proteins in total. Finally, we present the first deep-sequencing approach for negative mode proteomics, leveraging offline low-pH reversed-phase fractionation prior to online high-pH separations and peptide fragmentation with AI-NETD. With this platform, we identified 3,467 proteins in yeast with trypsin alone and characterized a total of 3,730 proteins using multiple proteases, or nearly 83% of the expressed yeast proteome. This work represents the most extensive negative mode proteomics study to date, establishing AI-NETD as a robust tool for large-scale peptide anion characterization and making the negative mode approach a more viable platform for future proteomic studies.
Project description:The structure of the Bowman-Birk inhibitor from Vigna unguiculata seeds (BTCI) in complex with beta-trypsin was solved and refined at 1.55 A to a crystallographic R(factor) of 0.154 and R(free) of 0.169, and represents the highest resolution for a Bowman-Birk inhibitor structure to date. The BTCI-trypsin interface is stabilized by hydrophobic contacts and hydrogen bonds, involving two waters and a polyethylene glycol molecule. The conformational rigidity of the reactive loop is characteristic of the specificity against trypsin, while hydrophobicity and conformational mobility of the antichymotryptic subdomain confer the self-association tendency, indicated by atomic force microscopy, of BTCI in complex and free form. When BTCI is in binary complexes, no significant differences in inhibition constants for producing a ternary complex with trypsin and chymotrypsin were detected. These results indicate that binary complexes present no conformational change in their reactive site for both enzymes confirming that these sites are structurally independent. The free chymotrypsin observed in the atomic force microscopy assays, when the ternary complex is obtained from BTCI-trypsin binary complex and chymotrypsin, could be related more to the self-association tendency between chymotrypsin molecules and the flexibility of the reactive site for this enzyme than to binding-related conformational changes.
Project description:Proteomic analysis of sensory organs such as the cochlea is challenging due to its small size and difficulties with membrane protein isolation. Mass spectrometry in conjunction with separation methods can provide a more comprehensive proteome, because of the ability to enrich protein samples, detect hydrophobic proteins, and identify low abundant proteins by reducing the proteome dynamic range. GELFrEE as well as different separation and digestion techniques were combined with FASP and nanoLC-MS/MS to obtain an in-depth proteome analysis of cochlear sensory epithelium from 30-day-old mice. Digestion with LysC/trypsin followed by SCX fractionation and multiple nanoLC-MS/MS analyses identified 3773 proteins with a 1% FDR. Of these, 694 protein IDs were in the plasmalemma. Protein IDs obtained by combining outcomes from GELFrEE/LysC/trypsin with GELFrEE/trypsin/trypsin generated 2779 proteins, of which 606 additional proteins were identified using the GELFrEE/LysC/trypsin approach. Combining results from the different techniques resulted in a total of 4620 IDs, including a number of previously unreported proteins. GO analyses showed high expression of binding and catalytic proteins as well as proteins associated with metabolism. The results show that the application of multiple techniques is needed to provide an exhaustive proteome of the cochlear sensory epithelium that includes many membrane proteins. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD000231.
Project description:1. A comparison of the diagonal ;maps' of chymotrypsin A and ;tosylphenylalanyl chloromethyl ketone'-inhibited chymotrypsin A showed that His-57 is alkylated specifically by this substrate analogue. 2. From peptic digests of chymotrypsinogen A and B, trypsin and elastase it was demonstrated by the diagonal electrophoretic technique that a common di-histidine cystine-bridged structure is present in all four enzymes. 3. The sequences of these peptides were determined and show that the positions of the two histidine residues relative to the disulphide bond are a common feature. Thus His-40 of chymotrypsin A is only two residues removed from CyS-42, and His-57 is adjacent to the other half of this bridge, CyS-58. 4. Considerable variation in sequence occurs about His-40, where the aromatic residues 39 and 41 of the chymotrypsins and trypsin are replaced by alanine and threonine in elastase. There is a remarkable similarity in sequence following CyS-42 and preceding CyS-58 in all four enzymes.
Project description:This study aimed to investigate the effects of leucine (Leu) on the synthesis and secretion of digestive enzymes in cultured pancreatic tissue of dairy goats and on the signaling molecules. Fresh pancreatic tissue from dairy goats was cut into approximately 2?mm?×?2?mm pieces and incubated in oxygenated Krebs-Ringer bicarbonate buffer containing 0 (the control), 0.40, 0.80, or 1.60?mM Leu at 39°C in a CO2 incubator for 180?min. The results showed that Leu increased the release of ?-amylase, trypsin, and chymotrypsin in the buffer and tissue, as well as the total activity (P < 0.05), especially at 0.40 and 0.80?mM. Compared with the control, 1.60?mM Leu increased the release of ?-amylase and the total activity of trypsin and chymotrypsin (P < 0.05) but had no effect on the tissue concentration of ?-amylase, trypsin, and chymotrypsin or the total activity of ?-amylase (P > 0.05). Leu improved the mRNA expression of ?-amylase, trypsin, and chymotrypsin (P < 0.05), especially at 0.80 and 1.60?mM. The activity and mRNA expression of lipase were not affected (P > 0.05). Compared with the control, 0.40 and 0.80?mM Leu increased the expression of the ? isoform of 4EBP1 (P < 0.05), implying increased phosphorylation of 4EBP1. Leu increased the phosphorylation of S6K1 (P < 0.05). Compared with the control, 0.40 and 0.80?mM Leu decreased the eEF2 phosphorylation level (P < 0.05). Conclusively, these results suggested that Leu could regulate the synthesis of pancreatic enzymes by increasing the mRNA expression and phosphorylation level of protein factors in the mammalian target of rapamycin pathway and the optimal Leu level in this experiment was 0.80?mM.