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)

Two chymotrypsin-like proteases were purified from the secretory and excretory material of first-instar larvae of Lucilia cuprina. The hydrolysis of N-succinyl-L-phenylalanine-nitroanilide was used to monitor the purification of these proteases which was achieved by affinity chromatography on soybean trypsin inhibitor-Sepharose followed by anion exchange and hydrophobic interaction chromatographies. The enzymatic specificity of the most abundant protease (Lucilia chymotrypsin b; LCTb) was further defined by determining the amino acid sequence of peptides released from insulin B chain after incubation with LCTb. Peptide amino acid sequences obtained from LCTb were used to design degenerate oligonucleotide primers which, in conjunction with the polymerase chain reaction, enabled cDNA coding for LCTb to be cloned and sequenced. The deduced amino acid sequence of LCTb showed many of the structural features of serine proteases as well as significant amino acid sequence homology with chymotrypsins from a diverse range of species. It is probable that LCTb plays an important role in establishing the myiasis-causing larvae of L. cuprina on host skin as well as providing nutrients for the rapidly growing larvae.
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PMID:Excretory/secretory chymotrypsin from Lucilia cuprina: purification, enzymatic specificity and amino acid sequence deduced from mRNA. 770 4

This report describes the cloning and sequencing of a novel protease gene derived from Streptomyces griseus. Also described is the heterologous expression of the gene in Bacillus subtilis and characterization of the gene product. The sprD gene encodes a prepro mature protease of 392 amino acids tentatively named S. griseus protease D (SGPD). A significant component of the enzyme preregion was found to be homologous with the mitochondrial import signal of hsp60. The sprD gene was subcloned into an Escherichia coli/B. subtilis shuttle vector system such that the pro mature portion of SGPD was fused in frame with the promoter, ribosome binding site, and signal sequences of subtilisin. The gene fusion was subsequently expressed in B. subtilis DB104, and active protease was purified. SGPD has a high degree of sequence homology to previously described S. griseus proteases A, B, C, and E and the alpha-lytic protease of Lysobacter enzymogenes, but unlike all previously characterized members of the chymotrypsin superfamily, the recombinant SGPD forms a stable alpha 2 dimer. The amino acid sequence of the protein in the region of the specificity pocket is similar to that of S. griseus proteases A, B, and C. The purified enzyme was found to have a primary specificity for large aliphatic or aromatic amino acids. Nucleotide sequence data were used to construct a phylogenetic tree using a method of maximum parsimony which reflects the relationships and potentially the lineage of the chymotrypsin-like proteases of S. griseus.
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PMID:Protease evolution in Streptomyces griseus. Discovery of a novel dimeric enzymes. 770 7

To characterize a chymotrypsin-like hydrolytic activity in the cell surface membranes of intact opossum kidney (OK) cells, we partially purified a protease from the membrane fractions of OK cells using Suc-Leu-Leu-Val-Tyr-MCA (Suc, succinyl; MCA, 4-methylcoumaryl-7-amide), a synthetic substrate for chymotrypsin, as the substrate. The semipure enzyme showed seryl chymotrypsin-like characteristics such as preferential hydrolysis of Suc-Leu-Leu-Val-Tyr-MCA and inhibition by phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, and chymostatin. However, it clearly differed from alpha-chymotrypsin in its weak ability to hydrolyze Suc-Ala-Ala-Pro-Phe-MCA and in its high molecular mass (250-300 kDa). The enzyme also had an endopeptidase-like activity in that it cleaved human parathyroid hormone(1-84) at the Leu(37)-Gly(38) and Arg(52)-Lys(53) bonds. These results suggest that a high molecular mass chymotrypsin-like endopeptidase with unique characters is present in the membrane fractions of OK cells.
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PMID:Characterization of a chymotrypsin-like hydrolytic activity in the opossum kidney cell. 781 50

Converting the specificity of trypsin to that of chymotrypsin has been shown to require the exchange of amino acids in multiple portions of the protein, including two surface loops which do not directly contact the substrate. Crystallographic analysis of two mutant trypsins possessing chymotrypsin-like specificity now reveals that these distal surface loops alter function by directly determining the structure of the primary binding site. Efficient acylation of cognate substrates correlates with a distinct backbone conformation of the conserved Gly216 residue. This amino acid is located on the surface of the specificity pocket and forms two main-chain hydrogen bonds with a nonspecific portion of substrate. By contrast, the improvement in substrate binding affinity effect by the substitution of the distal Tyr172 residue with Trp derives from structural rearrangements at the extreme base of the pocket. Together, the kinetic and crystallographic data strongly suggest that both Asp189 and Gly216 must be considered as primary determinants of substrate specificity in trypsin.
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PMID:Structural origins of substrate discrimination in trypsin and chymotrypsin. 784 8

Two fluorogenic N-nitrosoamides, N-nitroso-N-((7-methoxycoumarin-4-yl)methyl)-N'-isobutyrylalaninamide (6a) and N-nitroso-N-((6-methoxyquinolin-2-yl)methyl)-N'-isobutyrylalani namide (6b), were synthesized. Both N-nitrosoamides inhibited alpha-chymotrypsin irreversibly; they show promise as labeling reagents for the active sites of chymotrypsin-like proteases.
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PMID:Fluorogenic N-nitrosoamides: active-site labeling reagents for chymotrypsin-like proteases. 784 76

The polyprotein encoded by a single open reading frame of hepatitis C virus (HCV) is processed by host- and virus-encoded proteases. The viral protease NS3 is responsible for the cleavage of at least four sites (NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B junctions) in the nonstructural protein region. To characterize the protease function of NS3 and NS4 on various target sites, efficient cis- and trans-cleavage assay systems were developed by using in vitro transcription and translation. Deletion of the C-terminal two-thirds from NS3 in an NS3-NS4A-4B polypeptide (NS3 delta C-4A-4B) hampered cleavage of the NS3/4A junction but not that of the NS4A/4B junction. As a consequence, expression of NS3 delta C-4A-4B containing an internal deletion of NS3 results in an NS3 delta C-4A fusion protein. NS3 delta C-4A shows very efficient and specific trans-cleavage activity at NS4A/4B, NS4B/5A, and NS5A/5B junctions. In addition, the biochemical properties of HCV NS3 delta C-4A were further elucidated by adding known protease inhibitors in trans-cleavage reactions. The HCV protease NS3-4A is inhibited by chymotrypsin-specific inhibitors N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), chymostatin, and Pefabloc SC but not by trypsin-like protease inhibitors antipain, leupeptin, and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) or by the protease inhibitors E-64, bestatin, pepstatin, and phosphoramidon. This finding strongly suggests that HCV protease NS3-4A is a chymotrypsin-like serine protease.
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PMID:NS3-4A of hepatitis C virus is a chymotrypsin-like protease. 788 3

Various protease inhibitors active against both trypsin- and chymotrypsin-like serine proteases were used to characterize gut proteases from Lucilia cuprina by in vitro feeding assays. Significant larval growth retardation was observed on feeding first-instar larvae with trypsin inhibitors, particularly soybean trypsin inhibitor. Feeding of chymostatin, a specific chymotrypsin inhibitor, resulted in no significant growth retardation. This information suggests that trypsin-like serine proteases are probably the major gut digestive enzymes. A DNA fragment obtained by PCR which coded for part of a putative trypsin gene from L. cuprina was used to isolate a four-member multigene family of trypsins. The full nucleotide sequence of one of the genes and partial sequence from the other three genes were determined. Transcription of at least one of the genes has been confirmed. All four of the genes appear to have arisen by two separate gene duplication events.
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PMID:Isolation of a trypsin-like serine protease gene family from the sheep blowfly Lucilia cuprina. 789 48

An inhibitor of pancreatic elastase (EI), which can also inhibit chymotrypsin, and an inhibitor of trypsin (TI), which can also inhibit plasmin, have been isolated from bovine plasma. EI and TI belong to the serpin family of inhibitors. The size of both inhibitors is approx. 60 kDa and they are able to form SDS-stable complexes with proteinases. Curiously, TI dimerizes in the presence of SDS, a feature which has been observed previously only in non-denaturing gels of human alpha 1-antitrypsin (alpha 1PI). EI and TI are glycosylated [16% and 19% (w/w) respectively] and their amino acid compositions are similar to those of other plasma serpins. Neither EI nor TI is the equivalent of bovine alpha 1PI, as revealed by partial sequence analysis of their N-termini and reactive sites. Rather, both inhibitors appear to be related to human alpha 1-antichymotrypsin. Inhibition of pancreatic elastase and chymotrypsin by EI occurs with a kass. approximately 10(5) M-1.s-1. TI inhibits trypsin with a kass. approximately 10(5) M-1.s-1. Plasmin is inhibited by TI with a kass. approximately 10(3) M-1.s-1. The values of the kinetic constants are similar to those determined for the well-studied human serpins. Antibodies to EI and TI reveal a set of four antigenically related proteins of similar size in plasma. In addition, they detect the same set of proteins in milk. The inhibitors isolated from milk are identical to EI and TI from plasma. EI could control the activity of chymotrypsin-like proteinases in milk. In contrast, no target proteinases of TI in milk can be suggested.
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PMID:Characterization of two serpins from bovine plasma and milk. 798 Mar 96

Trypsin and chymotrypsin have very similar tertiary structures, yet very different substrate specificities. Recent site-directed mutagenesis studies have shown that mutation of the residues of the substrate binding pocket of trypsin to the analogous residues of chymotrypsin does not convert trypsin into a protease with chymotrypsin-like specificity. However, chymotrypsin-like substrate specificity is attained when two surface loops are changed to the analogous residues of chymotrypsin, in conjunction with the changes in the S1 binding site [Hedstrom, L., Szilagyi, L., & Rutter, W. J. (1992) Science 255, 1249-1253). This mutant enzyme, Tr-->Ch[S1+L1+L2], is improved to a protease with 2-15% of the activity of chymotrypsin by the mutation of Tyr172 to Trp. Residue 172 interacts synergistically with the residues of the substrate binding pocket and the loops to determine substrate specificity. Further, these trypsin mutants demonstrate that substrate specificity is determined by the rate of catalytic processing rather than by substrate binding.
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PMID:Converting trypsin to chymotrypsin: residue 172 is a substrate specificity determinant. 803 65

Rat trypsin II has been converted to a protease with chymotrypsin-like substrate specificity [Hedstrom, L., et al. (1994) Biochemistry (preceding paper in this issue)]. The key alteration in this conversion is the exchange of two surface loops for the analogous loops of chymotrypsin. k(inact)/Ki for the inactivation of chymotrypsin, trypsin, a trypsin mutant with poor activity (D189S), and the chymotrypsin-like mutants Tr-->Ch[S1+L1+L2] and Tr-->Ch[S1+L1+L2+Y172W] by Suc-Ala-Ala-Pro-Phe-chloromethylketone correlates with kcat/Km for hydrolysis of Suc-Ala-Ala-Pro-Phe-AMC. k(inact)'s for the inactivation of Tr-->Ch[S1+L1+L2] and Tr-->Ch[S1+L1+L2+Y172W] are comparable to that of chymotrypsin, while Ki's were much higher. Ki for the inhibition of these enzymes by the transition-state analog MeOSuc-Ala-Ala-Pro-boro-Phe also correlates with kcat/Km for hydrolysis of Suc-Ala-Ala-Pro-Phe-AMC. These results suggest that the surface loops stabilize the transition state for hydrolysis of chymotrypsin substrates by improving the orientation of bound substrates relative to the catalytic residues. Lastly, trypsin and chymotrypsin have comparable affinities for proflavin, while the Kd for the Tr-->Ch[S1+L1+L2+Y172W]-proflavin complex is 10-fold higher. No proflavin binding could be observed for either D189S or Tr-->Ch-[S1+L1+L2], which suggests that the S1 binding pockets of these two mutant enzymes are deformed. This work confirms that enzyme specificity is expressed in the chemical steps of the reaction rather than in substrate binding.
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PMID:Converting trypsin to chymotrypsin: ground-state binding does not determine substrate specificity. 803 66


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