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)

Crystal forms 2 and 3 of Sindbis virus core protein have been refined to 2.8 A and 3.0 A resolution, respectively. The three independent molecular copies in the two crystal forms are essentially identical, except for regions where the molecules are involved in different crystal packing interactions. The overall polypeptide backbone fold of Sindbis virus core protein is similar to other chymotrypsin-like serine proteinase structures despite a lack of significant sequence homology. Detailed analysis revealed differences in the catalytic triad and the substrate binding pockets between the Sindbis virus core protein and the other serine proteinases. The catalytic aspartic acid residue (Asp163) and residue Asp214 (corresponding to Asp194 in chymotrypsin) are partially exposed to solvent in Sindbis virus core protein. Chymotrypsin Ser214, hydrogen bonded to the catalytic aspartic acid residue in all other serine proteinase structures, is changed to Leu231 in Sindbis virus core protein. Deletions in the loop regions on the surface of the protein account for the smaller size of the ordered part of Sindbis virus core protein (151 residues) as compared to chymotrypsin (236 residues), and permits the cis autocatalytic cleavage of the polyprotein to produce the viral capsid protein.
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PMID:Refined structure of Sindbis virus core protein and comparison with other chymotrypsin-like serine proteinase structures. 845 May 38

E. coli L-asparaginase was modified with N,O-carboxymethyl chitosan in the presence of normal product L-aspartic acid, which protected the active site of the enzyme. The modified enzyme remained high catalytic activity, showed greater stability against trypsin and alpha-chymotrypsin, but lost its activity more rapidly at high temperature (> 45 degrees C) than did the native enzyme. When tested in vivo, the plasma half-life of the modified enzyme (t1/2 = 40 hr) was over 33 times longer than that of the native enzyme (t1/2 = 1.6 hr). The results showed that the modified L-asparaginase may be much more useful than did the native enzyme for clinical treatments of tumors.
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PMID:The chemical modification of E. coli L-asparaginase by N,O-carboxymethyl chitosan. 892 26

In order to understand the regulation of trypsin genes by the blood meal, we constructed a cDNA library from mRNA isolated from midguts of blood-fed female Aedes aegypti. The library was screened with a Drosophila melanogaster trypsin-like gene; twelve cDNAs were isolated and sequenced. Two clones were 846 bp and 788 bp long with 762 bp and 716 bp open reading frames, respectively. The cDNAs were identified as coding for serine proteases by the presence of conserved serine, histidine and aspartic acid residues; the presence of an aspartate residue at position 176 suggests that the clones were derived from trypsin-like gene transcripts rather than chymotrypsin or other serine proteases. One of the clones contained a 5' untranslated region and coding regions for putative signal and activation peptides, suggesting that the product is secreted as an inactive zymogen and processed by autoactivation. Southern analysis of genomic DNA suggests that trypsin is encoded by a multigene family in A. aegypti.
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PMID:Isolation, sequencing and characterization of two cDNA clones coding for trypsin-like enzymes from the midgut of Aedes aegypti. 908 45

Histone H1(0) is known to consist of two subfractions named H1(0)a and H1(0)b. The present work was performed with the aim of elucidating the nature of these two subfractions. By using reversed-phase high performance liquid chromatography in combination with hydrophilic interaction liquid chromatography, we fractionated human histone H1(0) into even four subfractions. Hydrophilic interaction liquid chromatographic analysis of the peptide fragments obtained after cleavage with cyanogen bromide and digestion with chymotrypsin suggested that the four H1(0) subfractions differ only in their small N-terminal end of the H1(0) molecule (30 residues). Edman degradation of the N-terminal H1(0) peptide fragments and mass spectra analysis have indicated that human histone H1(0) consists of intact histones H1(0) (named H1(0) Asn-3) and deamidated H1(0) forms (H1(0) Asp-3) having an aspartic acid residue at position 3 instead of asparagine. Moreover, both H1(0) Asn-3 and H1(0) Asp-3 are blocked (H1(0)a Asn-3, H1(0)a Asp-3) and unblocked (H1(0)b Asn-3, H1(0)b Asp-3) on their N terminus. Acid-urea gel electrophoretic analysis has shown that the histone subfraction, in the literature originally named H1(0)a, actually consists of a mixture of H1(0)a Asn-3 and H1(0)a Asp-3, whereas H1(0)b consists of H1(0)b Asn-3 and H1(0)b Asp-3. Furthermore, we found that hydrophilic interaction liquid chromatography separates rat and mouse histone H1(0) just like human H1(0) into four subfractions. Hydrophilic interaction liquid chromatographic analysis of brain and liver histone H1(0) from rats of different ages revealed an age-dependent increase of both the N-terminally acetylated and the deamidated forms of H1(0). In addition, we found that the relative proportions of the four forms of H1(0) histones differ from tissue to tissue.
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PMID:The microheterogeneity of the mammalian H1(0) histone. Evidence for an age-dependent deamidation. 958 79

Protein A (PA) of Staphylococcus aureus has an array of biological functions, such as antitumor, antitoxic, anticarcinogenic, immunomodulatory, antifungal, and antiparasitic properties. We have already established that a theoretical trypsin-digested peptide fragment of protein A (20-mer) mimics immunomodulatory and IgG binding property of PA. In the present report we have concentrated on a 16-mer chymotryptic fragment of protein A, which has a sequence of 13 amino acids in common with the previously studied 20-mer peptide. Molecular modeling study qualitatively predicted that both 20-mer and 16-mer peptides retain Fc binding ability from an interaction energy point of view. In the present study our aim was to understand whether this theoretically predicted 16-mer chymotryptic fragment could be formed in a real experiment and also to understand its biological activities. Chymotrypsin cleavage of PA at 37 degrees C for 24 h produced four major fragments on reverse-phase HPLC. The amino acid analyses of each fragment show the absence of cysteine residue from all fragments, which justifies the absence of cysteine in PA. We also observed high content of aspartic acid and glutamic acid residues in all fragments. On gel-filtration chromatography the chymotrypsin cleavage of PA shows five peaks, one of which overlaps with our theoretically selected 16-mer peptide on superimposition. We verified the IgG binding capacity of 16-mer peptide by capillary electrophoresis. The 16-mer peptide also induces the production of TNFalpha and IL-1alpha in serum of mice. The above observations suggest that the 16-mer peptide may be produced by chymotrypsin cleavage and also that this peptide possesses some of the major biological properties of PA, such as IgG binding, TNFalpha and IL-1alpha elicitation, etc.
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PMID:Functional mimicry of protein A of Staphylococcus aureus by a proteolytically cleaved fragment. 1038 52

Two genomic DNA fragments encoding crustacean collagenolytic serine protease genes show coding fragments that span 1522-1526 base pairs and contain seven exons encoding the complete amino acid sequence of two enzymes, CHYA and CHYB. As in serine protease genes from other organisms, the region coding for the residues around the active site is split by two introns. Although the introns differ from those of other organisms in size and nucleotide sequence, their number and location are more or less the same as found in mammalian chymotrypsin or elastase genes that evolved lately, but different for trypsin genes. Meanwhile, the junction that occurs between the propeptide and the maturation site is only found in the shrimp genes. This is also the case for the junction located 13 amino acids after the active site aspartic acid in these genes. Between 40 and 50 copies of the genes are reported by Southern analysis. Seven different genes within ChyA Pv family present 0-6% base changes, whereas five different genes belonging to ChyB Pv family show changes of up to 27% in the short studied portion of exon 4. This last family presents a mosaic organization of the coding parts, which are also expressed in the hepatopancreas of the shrimp as the variant PVC5 cDNA.
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PMID:Polymorphism and evolution of collagenolytic serine protease genes in crustaceans. 1040 65

Based on homology, amino acids 326-336 (143-154 in chymotrypsin numbering) of factor X (fX) comprise a flexible surface loop, which is susceptible to self-proteolysis and influences substrate catalysis. To investigate the role of this autolysis loop in fX function, a recombinant variant with a new site for asparagine-linked glycosylation has been produced by changing glutamine 333 to asparagine. Q333N fX is activated normally by factor VIIa and tissue factor, factors IXa and VIIIa, and Russell's viper venom. Proteolysis of the loop is prevented by the mutation. Reactivity of the free enzyme toward substrates and inhibitors is attenuated 4-20-fold; relative to wild type fXa, Spectrozyme Xa(TM) hydrolysis is 25%, inhibition by antithrombin III and the tissue factor pathway inhibitor is approximately 20%, and prothrombin activation in the absence of the cofactor Va is only 5%. Surprisingly, activities of the variant and wild type enzymes are equivalent when part of the prothrombinase complex. N-Glycanase cleaves the new oligosaccharide from Q333N fXa leaving aspartic acid. Q333D fXa is approximately 1.6-fold more reactive with Spectrozyme Xa(TM), antithrombin III and tissue factor pathway inhibitor, and prothrombin than its glycosylated counterpart, Q333N fXa, but still quite abnormal relative to wild type fXa. Like Q333N fXa, Q333D fXa is fully functional as part of the prothrombinase complex. We conclude that Gln-333 is geographically close to a site of proteolytic degradation but not to activator, cofactor, or membrane binding sites. Mutation of Gln-333 impairs catalytic function, but given normal prothrombin activation by the complexed enzyme, the importance of Gln-333 for catalysis is not manifest in the prothrombinase assembly, suggesting a conformational change in complexed fXa.
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PMID:Directed glycosylation of human coagulation factor X at residue 333. Insight into factor Va-dependent prothrombin catalysis. 1099 46

Nuclear inclusion a (NIa) protease of tobacco vein mottling virus is responsible for the processing of the viral polyprotein into functional proteins. In order to identify the active-site residues of the TVMV NIa protease, the putative active-site residues, His-46, Asp-81 and Cys-151, were mutated individually to generate H46R, H46A, D81E, D81N, C151S, and C151A, and their mutational effects on the proteolytic activities were examined. Proteolytic activity was completely abolished by the mutations of H46R, H46A, D81N, and C151A, suggesting that the three residues are crucial for catalysis. The mutation of D81E decreased kcat marginally by about 4.7-fold and increased Km by about 8-fold, suggesting that the aspartic acid at position 81 is important for substrate binding but can be substituted by glutamate without any significant decrease in catalysis. The replacement of Cys-151 by Ser to mimic the catalytic triad of chymotrypsin-like serine protease resulted in the drastic decrease in kcat by about 1,260-fold. This result might be due to the difference of the active-site geometry between the NIa protease and chymotrypsin. The protease exhibited a bell-shaped pH-dependent profile with a maximum activity approximately at pH 8.3 and with the abrupt changes at the respective pKa values of approximately 6.6 and 9.2, implying the involvement of a histidine residue in catalysis. Taken together, these results demonstrate that the three residues, His-46, Asp-81, and Cys-151, play a crucial role in catalysis of the TVMV NIa protease.
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PMID:Characterization of active-site residues of the NIa protease from tobacco vein mottling virus. 1110 Nov 40

Two new trypsin inhibitors, TDI-I and TDI-II, were purified from the seeds of the native Brazilian tree Copaifera langsdorffii (Caesalpinoideae, Leguminosae). The purification procedure involved ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Sepharose, affinity chromatography on trypsin-Sepharose, and reversed-phase (RP) HPLC. RP-HPLC yielded two forms (TDI-I and TDI-II), as confirmed by isoelectric focusing, with pI values between 7.0 and 8.1. The molecular mass of the TDI forms was 24 kDa based on FPLC gel filtration on Superdex 75. Under reducing conditions in tricine SDS-PAGE the molecular masses of TDI-I and TDI-II were 12 and 10 kDa, respectively. The Ki values were 1.1 and 1.2 nM for TDI-I and TDI-II, respectively, and there was no inhibitory effect on chymotrypsin. Amino acid analysis revealed high levels of aspartic acid, glutamic acid, serine, glycine, proline, and lysine but low levels of methionine and aromatic amino acids in both inhibitors; the calculated molecular masses were 11,456 and 10,008 for TDI-I and II, respectively. Based on the N-terminal sequences of TDI-I and TDI-II, TDI-I belongs to the Kunitz family of trypsin inhibitors, whereas TDI-II showed no homology to any other protein. This observation suggests that TDI-II belongs to a new inhibitor subclass of low-molecular mass proteins in the subfamily Caesalpinoideae.
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PMID:Biochemical characterization and N-terminal sequences of two new trypsin inhibitors from Copaifera langsdorffii seeds. 1133 Mar 43

N-benzyloxycarbonyl-L-aspartic acid ethyl ester-D-alanine amide, a derivative of alitame, was synthesized from a eutectic mixture of the substrates N-benzyloxycarbonyl-L-aspartic acid diethyl ester and D-alanine amide using alpha-chymotrypsin. The hydrophilic solvents DMSO and MEA were found to be the best adjuvants for formation of a eutectic substrate mixture. A low eutectic temperature of 27 degrees C was obtained for the substrate mixture containing 9% DMSO, 18% MEA, and 12% water. Under these conditions a conversion yield of 70.3% (mol/mol) was obtained at 37 degrees C. The optimum molar ratio of the acyl acceptor D-alanine amide and the acyl donor N-benzyloxycarbonyl-L-aspartic acid diethyl ester was 1:1.
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PMID:Solvent-free enzymatic synthesis of alitame precursor using eutectic substrate mixtures. 1133 43


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