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)

A human urine serine proteinase chymotrypsin like hydrolyzes the peptide bonds: Phe-Ser (kinin); Gly-Gly, Leu-Arg, Phe-Lys (neuropeptides) and Gln-Gln (substance P). Endopeptidase H2 hydrolyzes better oligopeptides with 4 to 18 aminoacid residues than larger peptides, it does not hydrolyzes kininogen or proenkephalin. The enzyme behaves as an oligoendopeptidase.
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PMID:Further characterization of endopeptidase H2 a serine proteinase from human urine. 128 11

Thrombin is a multifunctional serine proteinase that plays a key role in coagulation while exhibiting several other key cellular bioregulatory functions. The X-ray crystal structure of human alpha-thrombin was determined in its complex with the specific thrombin inhibitor D-Phe-Pro-Arg chloromethylketone (PPACK) using Patterson search methods and a search model derived from trypsinlike proteinases of known spatial structure (Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S.R., & Hofsteenge, J., 1989, EMBO J. 8, 3467-3475). The crystallographic refinement of the PPACK-thrombin model has now been completed at an R value of 0.156 (8 to 1.92 A); in particular, the amino- and the carboxy-termini of the thrombin A-chain are now defined and all side-chain atoms localized; only proline 37 was found to be in a cis-peptidyl conformation. The thrombin B-chain exhibits the characteristic polypeptide fold of trypsinlike serine proteinases; 195 residues occupy topologically equivalent positions with residues in bovine trypsin and 190 with those in bovine chymotrypsin with a root-mean-square (r.m.s.) deviation of 0.8 A for their alpha-carbon atoms. Most of the inserted residues constitute novel surface loops. A chymotrypsinogen numbering is suggested for thrombin based on the topological equivalences. The thrombin A-chain is arranged in a boomeranglike shape against the B-chain globule opposite to the active site; it resembles somewhat the propeptide of chymotrypsin(ogen) and is similarly not involved in substrate and inhibitor binding. Thrombin possesses an exceptionally large proportion of charged residues. The negatively and positively charged residues are not distributed uniformly over the whole molecule, but are clustered to form a sandwichlike electrostatic potential; in particular, two extended patches of mainly positively charged residues occur close to the carboxy-terminal B-chain helix (forming the presumed heparin-binding site) and on the surface of loop segment 70-80 (the fibrin[ogen] secondary binding exosite), respectively; the negatively charged residues are more clustered in the ringlike region between both poles, particularly around the active site. Several of the charged residues are involved in salt bridges; most are on the surface, but 10 charged protein groups form completely buried salt bridges and clusters. These electrostatic interactions play a particularly important role in the intrachain stabilization of the A-chain, in the coherence between the A- and the B-chain, and in the surface structure of the fibrin(ogen) secondary binding exosite (loop segment 67-80).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The refined 1.9-A X-ray crystal structure of D-Phe-Pro-Arg chloromethylketone-inhibited human alpha-thrombin: structure analysis, overall structure, electrostatic properties, detailed active-site geometry, and structure-function relationships. 130 49

Human and bovine alpha-thrombin cleaved at the B-chain by chymotrypsin generates catalytically competent zeta-thrombins, which are comprised of two noncovalently linked fragments: a 36-(human) or 49-(bovine) residue A-chain linked by a disulfide to B-chain residues B1-148 (zeta 1-thrombin) and B-chain residues B149-259 (zeta 2-thrombin). Human and bovine D-Phe-Pro-Arg-CH2-zeta- and PhMeSO2-zeta-thrombins were prepared by reaction of the active-site histidine (H-B43) and serine (S-B205) with PPACK and PMSF, respectively. Unfolding and dissociation of the noncovalently linked polypeptide chains of either human or bovine D-Phe-Pro-Arg-CH2-zeta- and PhMeSO2-zeta-thrombins in 4.5 M guanidine-HCl and refolding upon 30-fold dilution in 50 mM sodium phosphate buffer pH 6.5, 750 mM NaCl, 0.1% PEG resulted in biphasic generation of catalytic activity. The slow phase was eliminated in the presence of the competitive inhibitor benzamidine-HCl. Unfolding and refolding mixtures of the appropriate inactive precursors generated the active chimeric thrombins bovine zeta 1-thrombin:human zeta 2-thrombin and human zeta 1-thrombin:bovine zeta 2-thrombin. Human zeta 1-thrombin and zeta 2-thrombin were isolated, and, upon recombining, the isolated fragments refolded to generate catalytically competent zeta-thrombin with an active-site content, specific activity toward Chromozym-TH, and a specificity constant (kcat/Km) for FPA release from fibrinogen that were all within 60% of those of native alpha-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Catalytically competent human and bovine zeta-thrombin and chimeras generated from unfolded polypeptide chains. 130 87

The precursor of Leu-enkephalin, Z-L-TyrGlyGly-L-Phe-L-LeuOEt, was synthesized from amino acid derivatives with three proteinases without the protection of the side chain of L-Tyr. First, Z-GlyGlyOBut and Z-L-TyrGlyGlyOBut were synthesized in quite a high yield, 83% and 99%, in an aqueous/organic biphasic system by papain and alpha-chymotrypsin, respectively. Then, Z-L-Phe-L-LeuOEt was synthesized by thermolysin from Z-L-Phe and L-LeuOEt either in buffer or in a biphasic system; the yields were 95% and 100%, respectively. The synthesis of Z-L-TyrGlyGly-L-Phe-L-LeuOEt from Z-L-TyrGlyGly and L-Phe-L-LeuOEt was performed effectively by thermolysin immobilized on Amberlite XAD-7 in a buffer and in an aqueous/organic biphasic system, as well as in saturated ethyl acetate, while the yield was low in reactions by free thermolysin. In the reaction with the immobilized enzyme (IME) in saturated ethyl acetate, the maximum yield of the precursor of Leu-enkephalin was 68%. The reasons for effective synthesis with IME are: (1) higher concentration of L-Phe-L-LeuOEt inside support, which resulted in rising the rate of the synthesis reaction and protecting the competitive hydrolysis of Z-L-TyrGlyGly by thermolysin, (2) entrapment of the product inside the support where thermolysin could not act in the case of reaction in buffer, and (3) extraction of the product with the organic solvent in the case of reaction in a biphasic system or in saturated organic solvent.
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PMID:Enzymatic synthesis of the precursor of Leu-enkephalin in water-immiscible organic solvent systems. 136 23

Peptide synthesis was carried out in a variety of organic solvents with low contents of water. The enzyme was deposited on the support material, celite, from an aqueous buffer solution. After evaporation of the water the biocatalyst was suspended in the reaction mixtures. The chymotrypsin-catalyzed reaction between Z-Phe-OMe and Leu-NH2 was used as a model reaction. Under the conditions used ([Z-Phe-OMe]0 less than or equal to 40 mM, [Leu-NH2]0/([Z-Phe-OMe]0 = 1.5) the reaction was first order with respect to Z-Phe-OMe. Tris buffer, pH 7.8, was the best buffer to use in the preparation of the biocatalyst. In water-miscible solvents the reaction rate increased with increasing water content, but the final yield of peptide decreased due to the competing hydrolysis of Z-Phe-OMe. Among the water-miscible solvents, acetonitrile was the most suitable, giving 91% yield with 4% (by vol.) water. In water-immiscible solvents the reaction rate and the product distribution were little affected by water additions in the range between 0% and 2% (vol. %) in excess of water saturation. The reaction rates correlated well with the log P values of the solvent. The highest yield (93%) was obtained in ethyl acetate; in this solvent the reaction was also fast. Under most reaction conditions used the reaction product was stable; secondary hydrolysis of the peptide formed was normally negligible. The method presented is a combination of kinetically controlled peptide synthesis (giving high reaction rates) and thermodynamically controlled peptide synthesis (giving stable reaction products).
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PMID:Enzymatic peptide synthesis in organic media: a comparative study of water-miscible and water-immiscible solvent systems. 136 30

An enzymic transesterification was carried out in a continuously operated fixed bed reactor. The reaction system consisted of immobilized alpha-chymotrypsin (E.C. 3.4.21.1) catalysing the transfer of the L-phenylalanine radical from the racemic propyl ester to 1,4-butanediol, yielding L-phenylalanine 4-hydroxybutyl ester. The desired reaction was accompanied by alcoholysis due to the presence of 1-propanol liberated during the reaction and by hydrolysis of both the propyl and the hydroxybutyl ester. The problem of shifting pH during the reaction due to ester hydrolysis was overcome by adjusting the initial pH of the substrate feed solution appropriately in order to obtain a sufficiently high buffer capacity provided by the free amino group of the esters. Thus, it was possible to work with shifting pH, an obvious disadvantage for operating reactors of low backmixing for this kind of reaction system. The overall reaction scheme was characterized by the appearance of a maximum ester yield as a function of the operating time in case of batch reactors. Surprisingly, the yield was found to become constant as a function of space-time for continuous operation due to a steeper pH drop. The maximum productivity achieved with respect to the hydroxybutyl ester was about 65 mol d-1 l-1 referred to the catalyst volume.
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PMID:Transesterification of phenylalanine by means of chymotrypsin in a continuous fixed bed reactor. 136 87

Eukaryotic peptidyl-prolyl cis-trans isomerases (rotamases) fall into two classes, the cyclophilins inhibited by cyclosporin A and the FK506-binding proteins inhibited by the macrolide antibiotic FK506. In prokaryotes homologs of cyclophilins have been identified and found to have rotamase activity. Sequence similarities have been noted between FK506-binding proteins and gene products in a number of bacterial species, but whether these bacterial proteins have rotamase activity is not known. Using the polymerase chain reaction, we have cloned and sequenced a homolog of an FK506-binding protein from Neisseria meningitidis and expressed the gene product as a fusion protein with maltose-binding protein. The fusion protein was purified by affinity chromatography. By measuring the rate of chymotrypsin cleavage of the substrate succinyl-Ala-Ala-Pro-Phe p-nitroanilide, we found that the fusion protein had rotamase activity comparable to that of human FK506-binding protein. This rotamase activity was inhibited by FK506.
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PMID:Neisseria meningitidis encodes an FK506-inhibitable rotamase. 137 54

The cell-free haemolymph of the mollusc Octopus vulgaris inhibited the proteolytic activity of the thermolysin against the high-molecular-mass substrate hide powder azure. The purified inhibitor was a glycoprotein composed of two identical 180 kDa disulphide-linked subunits. In addition to the inhibition of the metalloproteinase thermolysin, the protein inhibited the serine proteinases human neutrophil elastase, pig pancreatic elastase, bovine chymotrypsin, bovine trypsin and the cysteine proteinase papain. A fraction of the proteinase-inhibitor complex resisted dissociation after denaturation indicating that some of the proteinase molecules became covalently bound. The nucleophile beta-aminopropionitrile decreased the covalent binding of proteinases to the Octopus vulgaris protein, suggesting that this interaction is mediated by an internal thiol ester; the reactivity and the amino acid sequence flanking the reactive residues of the putative thiol ester were consistent with this hypothesis. Bound trypsin remained active against the low-molecular-mass chromatogenic substrate H-D-Pro-Phe-Arg p-nitroanilide and was protected from inhibition by active-site-directed protein inhibitors of trypsin; however, the bound trypsin was readily inhibited by small synthetic inhibitors. This indicates that the inhibition of proteinases is accomplished by steric hindrance. The proteinase-inhibitory activity of this protein is characteristic of inhibition by mammalian alpha-macroglobulins and the presence of a putative thiol ester suggests that the Octopus vulgaris proteinase inhibitor is a homologue of human alpha 2-macroglobulin.
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PMID:Purification and characterization of an alpha-macroglobulin proteinase inhibitor from the mollusc Octopus vulgaris. 137 44

Loss of chymotrypsin binding capacity of alpha 2-macroglobulin in diabetic plasma on in vitro incubation, could be partially prevented by phenylmethyl sulphonyl fluoride and pepstatin A. Prior ten-fold dilution of plasma with 0.02 M phosphate buffer (pH 7.0) completely arrested the process. The phenomenon could not be reactivated by Ca2+, lecithin or bovine serum albumin. Diabetic plasma, like normal plasma, exhibited maximal hydrolytic activities on H-D-Pro-Phe-Arg-p-nitroanilide, H-D-Val-Leu-Arg-p-nitroanilide and H-D-Ile-Pro-Arg-p-nitroanilide. The hydrolytic activities were not significantly diminished on incubation of plasma at 37 degrees C for 12 hr, unlike alpha 2-macroglobulin activity. On gel chromatography on Sephadex G-200, part of the proteolytic activity in diabetic plasma coeluted with alpha 2-macroglobulin in the VO region. A second activity peak (absent in normal plasma) was eluted with a Ve/V0 value of 1.40. Possible role of free proteinases in diabetic plasma in the inactivation of alpha 2-macroglobulin is discussed.
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PMID:Further studies on proteinases and alpha 2-macroglobulin activity in diabetic plasma. 138 30

Arachin, the major protein from groundnut, was isolated from three varieties of groundnut (Spanish Improved, TMV-2 and DH-3-30) using a modified procedure involving precipitation with 18% ammonium sulphate to obtain homogeneous protein. The homogeneity was judged by polyacrylamide gel electrophoresis, gel filtration and sedimentation velocity techniques as well as correlation with amino acid composition. Rates of hydrolysis of arachins by trypsin (pH 7.6) and alpha-chymotrypsin (pH 7.8) were significantly different between the three varieties. Arachin from the Spanish Improved variety contained higher amounts of alanine and phenylalanine and lower amounts of carbohydrate and phosphorus as compared to TMV-2 and DH-3-30. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis pattern of arachin from TMV-2 showed only seven bands of which the ones with low molecular weight were more intense than those of the other two varieties. The far-ultraviolet circular dichroic spectra showed no significant differences among the three varieties in respect of alpha-helix content (5 +/- 2%), beta-structure (19 +/- 2%) and the aperiodic structure. The observed differences in hydrolysis rates have been explained as due to the differences in the acidic and basic subunits of arachins.
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PMID:Effect of different proteolytic enzymes on the nature of subunit composition of arachins from groundnut (Arachis hypogaea L.). 139 8


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