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)

A series of N-acetyl-L-phenylalanyl peptides of general formula Ac-Phe-(Gly)n-NH2 (n = 0-2) has been synthesized to study the effect of leaving group chain length on the efficiency of chymotrypsin A alpha amidase and peptidase activities. The effect upon catalysis of hydrophobic side chains on the leaving group was investigated using similar substrates with one of the glycine residues selectively substituted by an alanine residue as in Ac-Phe-Ala-NH2, Ac-Phe-Ala-Gly-NH2, and Ac-Phe-Gly-Ala-NH2. Values of kcat and Km have been obtained from kinetic measurements at pH 8.00 and 25 degrees C. The results are shown to be consistent with binding schemes postulated from published model building studies. The catalytic reactions were studied over a range of temperature (15-35 degrees C) and in each case the Arrhenius law was obeyed. It was thus possible to obtain meaningful values for the thermodynamic functions of activation for the acylation step of the catalytic reaction. The results are shown to confirm the findings of postulated binding schemes but indicate that conclusions drawn from kinetic measurements at a single temperature may sometimes be misleading.
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PMID:Kinetic studies on the chymotrypsin A alpha-catalyzed hydrolysis of a series of N-acetyl-L-phenylalanyl peptides. 634 Jun 7

The amino acid sequence of deoxycytidylate deaminase isolated from T2 phage-infected Escherichia coli has been determined. The enzyme is a hexamer, consisting of identical polypeptide subunits, each composed of 188 amino acids with a calculated Mr = 20,560. The primary structure was established by automatic Edman degradation of the intact carboxymethylated protein and of peptides derived from the protein by cleavage with cyanogen bromide, trypsin, chymotrypsin, the Staphylococcus aureus V8 protease, and 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine. Knowledge of the primary structure of deoxycytidylate deaminase should aid in determining the allosteric binding site of the negative effector, dTTP, recently reported (Maley, F., and Maley, G.F. (1982) J. Biol. Chem. 257, 11876-11878), and eventually that of the enzyme's positive regulator, dCTP, as well as its substrate. The deaminase has been crystallized through the use of polyethylene glycol; a scanning electron micrograph is presented.
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PMID:Complete amino acid sequence of an allosteric enzyme, T2 bacteriophage deoxycytidylate deaminase. 634 41

The collagenolytic serine protease (crab protease) isolated from the hepatopancreas of the fiddler crab, Uca pugilator, has been investigated with respect to its peptide bond specificity and catalytic properties by using noncollagenous substrates. In contrast to vertebrate collagenases, crab protease is a good general protease capable of degrading a variety of polypeptide and synthetic low molecular weight substrates. Crab protease displays a broad range of specificity, cleaving on the carboxyl-terminal side of residues with both positively and negatively charged side chains as well as hydrophobic side chains. The enzyme appears to favor tyrosyl, phenylalanyl, leucyl, and perhaps lysyl residues and, to a lesser extent, arginyl and glutamyl residues. The rate of cleavage of polypeptide substrates is similar to chymotrypsin but is significantly less than trypsin or chymotrypsin for low molecular weight esterase and amidase substrates. Crab protease is effectively inhibited by chymostatin but not by leupeptin or elastatinal. Several common chloromethyl ketone derivatives of phenylalanine and lysine are also ineffective, although crab protease efficiently cleaves at these residues in polypeptide substrates.
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PMID:Substrate specificity of the collagenolytic serine protease from Uca pugilator: studies with noncollagenous substrates. 678 31

1. Two trypsin-like enzymes, assayed by their amidase activity with N-alpha-benzoyl-DL-arginine-p-nitroanilide (DL-BAPNA) as the substrate, were isolated from the gut of the arctic fish capelin (Mallotus villosus). 2. Purification involved affinity chromatography (Benzamidine-CH-Sepharose 4B) of the 30 to 70% (NH4)2SO4 precipitation fraction of a crude extract of the gut, followed by DEAE-Sephadex chromatography, yielding two enzymes, designated Enzyme I and II. 3. Both enzymes had MW of about 28,000 as determined by SDS-electrophoresis. Their isoelectric points were 5.6-5.9 (Enzyme I) and 5.1-5.3 (Enzyme II) and they had similar amino acid composition. 4. Both enzymes were inhibited by standard trypsin inhibitors including the serine protease inhibitor phenylmethyl sulphonyl fluoride (PMSF), but not by the chymotrypsin inhibitor L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK). 5. The enzymes had a pH optimum of 8-9 and their stability was not affected by CaCl2. Low pH (2.3) caused an initial rapid loss of enzyme activity, followed by relatively slow decomposition of the activity remaining after 1 hr at 4 degrees C. 6. The enzymes had an apparent temperature optimum of 42 degrees C, resulting from rapid self digestion at higher temperatures.
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PMID:Characteristics of two trypsin type isozymes isolated from the arctic fish capelin (Mallotus villosus). 708 13

Formiminotransferase-cyclodeaminase, an octameric protein of identical, bifunctional polypeptides of Mr = 62,000, yields a transferase-active fragment of Mr = 80,000 upon proteolysis with chymotrypsin in the presence of the inhibitor folic acid. The purified fragment contains one size of polypeptide, Mr = 39,000, on dodecyl sulfate gels. Cross-linking with the bifunctional reagent dithiobis(succinimidyl propionate) confirmed the dimeric structure of the purified fragment. Reaction of the native octamer with the very short bifunctional reagent difluorodinitrobenzene yields dimer and tetramer in excess of trimer, thereby indicating two types of subunit interaction in the protein. The isolation of a dimeric fragment after proteolysis and the results of cross-linking support a tetramer of dimers structure for the native enzyme. The purified transferase fragment has approximately 68% of the activity of the native enzyme, but has lost specificity for the naturally occurring polyglutamate derivatives of tetrahydrofolate. This is illustrated by an increase in Km for tetrahydropteroylpentaglutamate from 3.4 microM with the native transferase to 89 microM with the fragment transferase. It is suggested that the bifunctional enzyme may have only one polyglutamate binding site/pair of transferase-deaminase sites.
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PMID:The bifunctional enzyme formiminotransferase-cyclodeaminase is a tetramer of dimers. 741 Apr 36

A mouse monoclonal antibody against boar acrosin and antiserum prepared to highly purified acrosin in female rabbits were used to detect the antigen in various fluids and tissues of boars using an indirect immunofluorescence technique. A strong reaction was found in fluid and epithelial tissue of the seminal vesicles as well as in the germinal cells in the testis. No immunoreactivity was detected in tissues of the epididymides and other organs of the boar. The antigens present in seminal vesicle fluid of boars were partially purified by column chromatography. It was demonstrated that two antigens differing in molecular mass were present and both possessed protease and amidase activity. The higher molecular mass antigen eluted from a gel filtration column in a volume identical to that of proacrosin. The same result was obtained in polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE). The low molecular mass antigen was eluted from Sephadex G-75 column together with natural protease inhibitors corresponding in molecular mass to less than 20 kDa. The mobility of the antigen in SDS-PAGE was greater than that of chymotrypsin. It is assumed that the protease from seminal vesicle epithelial resembled acrosin in structure and function. Acrosin may therefore not be specific for spermatozoa.
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PMID:Serine protease activity in boar seminal vesicles and its immunological similarity to sperm acrosin. 802 64

Purified human serum butyrylcholinesterase, which exhibits cholinesterase, aryl acylamidase, and peptidase activities, was cross-reacted with two different monoclonal antibodies raised against human serum butyrylcholinesterase. All three activities were immunoprecipitable at different dilutions of the two monoclonal antibodies. At the highest concentration of the antibodies used, nearly 100% of all three activities were precipitated, and could be recovered to 90-95% in the immunoprecipitate. The peptidase activity exhibited by the purified butyrylcholinesterase was further characterized using both Phe-Leu and Leu-enkephalin as substrates. The pH optimum of the peptidase was in the range of 7.5-9.5 and the divalent cations Co2+, Mn2+, and Zn2+ stimulated its activity. EDTA and other metal complexing agents inhibited its activity. Thiol agents and -SH group modifiers had no effect. The serine protease inhibitors, diisopropylfluorophosphate and phenyl methyl sulfonyl fluoride, did not inhibit. When histidine residues in the enzyme were modified by diethylpyrocarbonate, the peptidase activity was not affected, but the stimulatory effect of Co2+, Mn2+, and Zn2+ disappeared, suggesting the involvement of histidine residues in metal ion binding. These general characteristics of the peptidase activity were also exhibited by a 50 kD fragment obtained by limited alpha-chymotrypsin digestion of purified butyrylcholinesterase. Under all assay conditions, the peptidase released the two amino acids, leucine and phenylalanine, from the carboxy terminus of Leu-enkephalin as verified by paper chromatography and HPLC analysis. The results suggested that the peptidase behaved like a serine, cysteine, thiol-independent metallopeptidase.
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PMID:The peptidase activity of human serum butyrylcholinesterase: studies using monoclonal antibodies and characterization of the peptidase. 842 27

Anandamide amidase (EC 3.5.1.4) is responsible for the hydrolysis of arachidonoyl ethanolamide (anandamide). Relatively selective and potent enzyme reversible inhibitors effective in the low micromolar range, such as arachidonyl trifluoromethyl ketone (Arach-CF3), have been described (Koutek et al., J Biol Chem 269: 22937-22940, 1994). In the current study, methyl arachidonyl fluorophosphonate (MAFP), an arachidonyl binding site directed phosphonylation reagent, was tested as an inhibitor of anandamide amidase and as a ligand for the CB1 cannabinoid receptor. MAFP was 800 times more potent than Arach-CF3 and phenylmethylsulfonyl fluoride (PMSF) as an amidase inhibitor in rat brain homogenates. In intact neuroblastoma cells, MAFP was also approximately 1000-fold more potent than Arach-CF3. MAFP demonstrated selectivity towards anandamide amidase for which it was approximately 3000 and 30,000-fold more potent than it was towards chymotrypsin and trypsin, respectively. MAFP displaced [3H]CP-55940 binding to the CB1 cannabinoid receptor with an IC50 of 20 nM vs 40 nM for anandamide. It bound irreversibly and prevented subsequent binding of the cannabinoid radioligand [3H]CP-55940 at that locus. These studies suggest that MAFP is a potent and specific inhibitor of anandamide amidase and, in addition, can interact with the cannabinoid receptors at the cannabinoid binding site. This is the first report of a potent and relatively selective irreversible inhibitor of arachidonoyl ethanolamide amidase.
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PMID:Methyl arachidonyl fluorophosphonate: a potent irreversible inhibitor of anandamide amidase. 906 28

Previous investigation [Tsui et al. (1996) Biochim. Biophys. Acta 1269: 41-46] showed that two active forms of alcohol dehydrogenase can be purified from grass carp. The use of a protease inhibitor and the results of SDS-PAGE analysis of the enzymes suggest that one form (ADH-C) is a proteolytic product of the other (ADH-I). In this study, the protease responsible for the cleavage was purified. The cleavage enzyme had a subunit molecular weight of 28 kDa. An inhibitor study identified it as a serine protease. It exhibited a strong chymotrypsin activity in both esterase and amidase assays with a pH optimum in the range 7.5-8.5. The purified chymotrypsin also cleaved the intact grass carp ADH-I into the two-fragment ADH-C, with an accompanying increase in enzyme activity. A similar effect was not found using horse liver alcohol dehydrogenase.
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PMID:Identification of an "alcohol dehydrogenase-activating" protease in grass carp hepatopancreas as a chymotrypsin. 944 19

The conversion of trypsin into a protease with chymotrypsin-like activity and specificity required substitution of fifteen residues in the S1 site and two surface loops with their chymotrypsin counterparts [Hedstrom,L., Szilagyi,L. and Rutter,W.J. (1992) Science, 255, 1249-1253]. These residues may define a set of general structural determinants of specificity in the trypsin family. In order to test this hypothesis, we have attempted to convert trypsin into a protease with specificity for substrates containing small aliphatic residues by replacing the S1 site and these surface loops with the analogous residues of elastase. Five elastase-like mutant enzymes were constructed with various combinations of these substitutions. Four mutant enzymes catalyze the hydrolysis of MeOSuc-Ala-Ala-Pro-Ala-SBzl more efficiently than the hydrolysis of Suc-Ala-Ala-Pro-Phe-SBzl. This observation indicates that the mutant enzymes have elastase-like esterase specificity. The best mutant, Tr-->E1-2, is a more specific esterase than elastase: the ratio of the values of kcat/Km for MeOSuc-Ala-Ala-Pro-Ala-SBzl and Suc-Ala-Ala-Pro-Phe-SBzl is greater than 160 for Tr-->E1-2 and 50 for elastase. However, the esterase activity of Tr-->E1-2 is 300-fold less than elastase; in addition, Tr-->E1-2 has no measurable amidase activity. Thus these substitutions do not construct a protease with elastase-like activity. These experiments indicate that a unique structural solution is required for each different specificity. Previous work suggested that instability of the S1 site is a major barrier to redesigning the specificity of trypsin. This view is corroborated by preliminary structural studies of Tr-->E1-2. One dimensional 1H NMR spectrum of Tr-->E1-2 suggests that the S1 site and the two surface loops of this mutant trypsin may be disordered.
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PMID:Converting trypsin to elastase: substitution of the S1 site and adjacent loops reconstitutes esterase specificity but not amidase activity. 974 19


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