Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

D-Alanine carboxypeptidase (CPase), a detergent-soluble penicillin-sensitive membrane enzyme of Bacillus stearothermophilus, Mr = 46,500, was digested with either trypsin or alpha-chymotrypsin to yield water-soluble fragments, designated T-CPase and Chy-CPase, respectively, each of Mr = approximately 45,000. These fragments were generated and purified in milligram quantities by digestion of CPase covalently immobilized on a penicillin affinity column. They retained full enzymatic activity, became significantly more resistant to thermal inactivation, and lost micellar detergent binding upon proteolysis. Each was derived from CPase by loss of a COOH-terminal hydrophobic peptide. CPase was reconstituted into bacterial lipid vesicles in an enzymatically active form. Penicillin-binding sites were equally distributed on both sides of the lipid bilayer, suggesting a random orientation of the CPase molecules. Neither T-CPase nor Chy-CPase reconstituted into lipid vesicles when treated in an identical manner. CPase was slowly cleaved from the surface of these vesicles by either trypsin or alpha-chymotrypsin, yielding T-CPase and Chy-CPase, respectively. These results demonstrate that CPase is comprised of a water-soluble catalytic domain and a COOH-terminal hydrophobic region which mediates the anchoring of this enzyme to the bacterial membrane.
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PMID:Cleavage of a COOH-terminal hydrophobic region from D-alanine carboxypeptidase, a penicillin-sensitive bacterial membrane enzyme. Characterization of active, water-soluble fragments. 43 18

D-Alanine carboxypeptidase is a penicillin-sensitive intrinsic membrane enzyme which is composed of a hydrophilic NH2-terminal catalytic domain (Mr congruent to 45,000 to 47,000) and a COOH-terminal membranous segment (approximately 20 to 30 amino acids in length) (Waxman, D. J., and Strominger, J. L. (1979) J. Biol. Chem. 254, 4863-4875; Waxman, D. J., and Strominger, J. L. (1981) J. Biol. Chem. 256, 2059-2066). The primary structures of the COOH-terminal 30 amino acids of two D-alanine carboxypeptidase purified from bacterial membranes were determined (residues numbered from the COOH terminus): Bacillus stearothermophilus: (formula see text) Water-soluble fragments of the B. stearothermophilus D-alanine carboxypeptidase were shown to be formed by cleavage after Phe27 or after Leu25 as indicated by carboxypeptidase A and B analysis and by the release of the four COOH-terminal chymotryptic peptides (Val26-Leu25, Ser24-Phe16, Val15-Trp12, and Thr11-Leu1) upon formation of water-soluble chymotrypsin D-alanine carboxypeptidase. This indicates that the membranous fragment is largely contained within the COOH-terminal 24 residues. Thus, this bacterial membrane protein probably does not contain the significant cytoplasmic domain characteristic of transmembrane proteins such as glycophorin. The absence of an uninterrupted stretch of 20 to 25 uncharged residues suggests that the membrane anchoring of D-alanine carboxypeptidase may differ from that of simple transmembrane proteins. Possible structures for the membranous segment of D-alanine carboxypeptidase are discussed.
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PMID:Primary structure of the COOH-terminal membranous segment of a penicillin-sensitive enzyme purified from two Bacilli. 678 May 59

The S' subsite specificity of four homologous serine proteases, rat chymotrypsin, rat trypsin, alpha-lytic protease, and cercarial protease from Schistosoma mansoni, was studied by measuring acyl-transfer reactions to 100 pentapeptide nucleophiles. Peptides of the general structures H-Xaa-Ala-Ala-Ala-Ala-NH2, H-Ala-Xaa-Ala-Ala-Ala-NH2, and H-Ala-Ala-Xaa-Ala-Ala-NH2 were synthesized, where Xaa is D-Ala, Cit, and all natural amino acids except Cys. The variable residues of these nucleophiles occupy the P'1, P'2, and P'3 positions in acyl-transfer reactions. The P'1 and P'2 residues were found to influence the efficiency of the nucleophiles by more than 2 orders of magnitude, whereas the S'3 subsite shows a lower specificity in all four enzymes. We synthesized consensus peptides of the general structure H-aa1-aa2-aa3-Ala-Ala-NH2, in which two or three positions were occupied by amino acids that showed the highest specificity in the first series of nucleophiles. Peptides with optimal amino acid residues in the P'2 and P'3 positions show a very high efficiency in chymotrypsin- and trypsin-catalyzed reactions. Otherwise, large specific side chains in the P'1 and P'3 positions of the nucleophiles show less than additive binding contributions due to steric hindrance. Comparison of chymotrypsin-catalyzed acyl-transfer reactions to nucleophiles of the structures H-Xaa-Leu-Arg-Ala-Ala-NH2 and H-Xaa-Ala-Ala-Ala-Ala-NH2 reveals a significantly different P'1 specificity for both series which confirms steric hindrance between large P'1 and P'3 residues.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Role of the S' subsites in serine protease catalysis. Active-site mapping of rat chymotrypsin, rat trypsin, alpha-lytic protease, and cercarial protease from Schistosoma mansoni. 815 42

We have developed a rapid and convenient procedure for the characterization of the S' subsite specificity of serine proteases. A mixture of peptide nucleophiles is incubated with the enzyme in the presence of excess of a specific ester substrate. The decrease in each nucleophile concentration is monitored by high-performance liquid chromatography analysis of the dansylated mixture. Relative kinetic parameters for each nucleophile in the mixture are then calculated using a new statistical algorithm that relates all pairs of nucleophiles. As a first application, we investigated the S'1 subsite specificity of chymotrypsin, trypsin, and a recently described trypsin mutant, Tr-->Ch[S1 + L1 + L2] with chymotrypsin-like primary specificity [Hedstrom, L., Szilagyi, L., & Rutter, W. J. (1992) Science 255, 1249-1253]. For this purpose 21 peptide nucleophiles of the general structure H-Xaa-Ala-Ala-Ala-Ala-NH2 were prepared by multiple solid-phase synthesis, where Xaa represents D-alanine, citrulline, and all natural amino acids except cysteine. Relative second-order rate constants for the enzyme-catalyzed acyl transfer to these nucleophiles were determined over a range of 10(2). Chymotrypsin and trypsin have markedly different S'1 specificities. The order of preference in chymotrypsin-catalyzed acyl transfer reactions is positively charged > aliphatic > aromatic >> negatively charged, D-Ala, Pro P'1 side chain. Trypsin prefers hydrophobic residues, but like chymotrypsin aliphatic residues are better than aromatic residues in P'1 position. The S'1 specificity of the mutant Tr-->Ch[S1 + L1 + L2] is similar to the specificity of trypsin; however, P'1 aromatic residues have low reactivity characteristic of chymotrypsin.
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PMID:Mapping the S' subsites of serine proteases using acyl transfer to mixtures of peptide nucleophiles. 847 65

New affine sorbents were synthesized involving tripeptide morpholides H-Ala-Ala-Leu-Mrp and H-D-Ala-Leu-Arg-Mrp as ligands that mimic substrates of subtilisin-like proteases and kallikrein, respectively. These were used for the isolation and purification of several proteases: trypsin, pepsin, alpha-chymotrypsin, thrombin, kallikrein, and termitase and were also efficient in the isolation of proteolytic enzymes from complex mixtures, such as the urine of children suffering from glomerulonephritis, hepatopancreas of Kamchatka crab, and dandelion roots. The ligands are competitive inhibitors of a number of proteases, and therefore, they were supposed to interact with the substrate binding sites in these enzymes.
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PMID:[Affinity sorbents with tripeptide morpholine ligands for isolation of proteases]. 951 26

The metabolism of three opioid tetrapeptides, Tyr-D-Arg-Phe-Nva-NH2, Tyr-D-Arg-Phe-Phe-NH2 and Tyr-D-Ala-Phe-Phe-NH2, was investigated in the presence of pure pancreatic enzymes (trypsin, chymotrypsin, elastase, carboxypeptidase A and carboxypeptidase B), as well as in the presence of pure carboxylesterase and aminopeptidase N. The cleavage patterns of the pure pancreatic enzymes were then compared with those found in rat and human jejunal fluid. Metabolism was also studied in homogenates from different intestinal regions (duodenum, jejunum, ileum and colon) and in enterocyte cytosol from rats. The effect of various protease inhibitors was investigated in the jejunal homogenate. The parent peptides were assayed by high-performance liquid chromatography and metabolites were identified by means of liquid chromatography-mass spectrometry. Of the pure enzymes, the quickest hydrolysis of the peptides was observed for the pancreatic enzymes chymotrypsin, trypsin and carboxypeptidase A. In most cases they formed the corresponding deamidated tetrapeptides (chymotrypsin and trypsin) or tripeptides with a missing C-terminal amino acid (carboxypeptidase A). Regional differences in intestinal metabolism rates were found for all three peptides (P < 0.001), with the highest rates observed in jejunal and/or colonic homogenates. The deamidated tetrapeptides were formed both in rat intestinal homogenates and in enterocyte cytosol. Metabolism in the jejunal homogenate was markedly inhibited by some serine and combined serine and cysteine protease inhibitors. In conclusion, the C-terminal amide of these tetrapeptides did not fully stabilise them against intestinal deamidase and carboxypeptidase activities. The significant hydrolysis of the peptides by pure chymotrypsin, trypsin and carboxypeptidase A showed that lumenal pancreatic proteases might be a clear metabolic obstacle in oral delivery even for small peptides such as these tetrapeptides.
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PMID:Investigations of the in-vitro metabolism of three opioid tetrapeptides by pancreatic and intestinal enzymes. 1093 29

A mouse mu opioid receptor was engineered to contain a FLAG epitope at the amino-terminus and a hexahistidine tag at the carboxyl-terminus to facilitate purification. Selection of transfected human embryonic kidney (HEK) 293 cells yielded a cell line that expressed the receptor with a B(max) of 10 pmol/mg protein. 3[H]Bremazocine exhibited high affinity binding to the epitope-tagged mu opioid receptor with a KD of 1.0 nM. The agonists [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO), morphine and [D-Ala(2),D-Leu(5)]enkephalin (DADL) competitively inhibited bremazocine binding to the tagged mu receptor with KI's of 3.5, 17 and 70 nM, respectively. Chronic treatment of cells expressing the epitope-tagged mu receptor with DAMGO resulted in down-regulation of the receptor, indicating that the tagged receptor retained the capacity to mediate signal transduction. The mu receptor was solubilized from HEK 293 cell membranes with n-dodecyl-beta-D-maltoside in an active form that maintained high affinity bremazocine binding. Sequential use of wheat germ agglutinin (WGA)-agarose chromatography, Sephacryl S300 gel filtration chromatography, immobilized metal affinity chromatography, immunoaffinity chromatography, and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) permitted purification of the receptor. The purified mu opioid receptor was a glycoprotein that migrated on SDS/PAGE with an apparent molecular mass of 80 kDa. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was used to identify and characterize peptides derived from the mu opioid receptor following in-gel digestion with trypsin or chymotrypsin, and precursor-derived tandem mass spectrometry (ms/ms) confirmed the identity of several peptides derived from enzymatic digestion of the mu opioid receptor.
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PMID:Purification and mass spectrometric analysis of the mu opioid receptor. 1455 61

In order to extend the use of proteases to organic synthesis and seek the rules of enzymatic reactions in organic media, we focused on unnatural substrates for proteases to form amide bonds. In this paper, the study of unnatural substrates containing D-amino acid residue, which act as acyl acceptors as well as acyl donors for proteases in organic media, is reported. Dermorphin is a heptapeptide (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH(2)) with potent analgesic activity. The N-terminal tetrapeptide is the minimum sequence that retains dermorphin activity, and is selected as the model compound in our study. Two dermorphin-(1-4) derivatives, Boc-Tyr-D-Ala-Phe-Gly-N(2)H(2)Ph and Boc-Tyr-D-Ala-Phe-Gly-NH(2), which contained a d-amino acid residue, were synthesized by proteases in organic media for the first time. The synthesis of these two dermorphin-(1-4) derivatives could be catalyzed by subtilisin with Boc-Tyr-D-Ala-OCH(2)CF(3) as an acyl donor substrate in AcOEt. The synthesis of dermorphin-(1-2) derivative Boc-Tyr-D-Ala-N(2)H(2)Ph was catalyzed by alpha-chymotrypsin in different organic solvents and D-Ala-N(2)H(2)Ph was used as an acyl acceptor substrate. Factors influencing the above enzymatic reactions were systematically studied.
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PMID:Synthesis of dermorphin-(1-4) derivatives catalyzed by proteases in organic solvents. 1568 45

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