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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Thrombin Quick II is one of two dysfunctional forms of thrombin derived from the previously described congenital dysprothrombin prothrombin Quick. Thrombin Quick II does not clot fibrinogen, hydrolyze p-nitroanilide substrates of thrombin, or bind N2-[5-(dimethylamino)naphthalene-1-sulfonyl]arginine N,N-(3-ethyl-1,5-pentanediyl)amide, a high-affinity competitive inhibitor of thrombin. To determine the structural alteration in thrombin Quick II, the reduced, carboxymethylated protein was hydrolyzed by a lysyl endopeptidase. A peptide not present in a parallel thrombin hydrolysate was identified by reverse-phase chromatography. The peptide was purified by rechromatography and subjected to Edman degradation which showed that Gly-558 of human prothrombin had been replaced by Val. This corresponds to a point mutation of the Gly codon GGC to GUC. This Gly residue, which is highly conserved in the chymotrypsin family of serine proteases, forms part of the substrate binding pocket for bulky aromatic and basic side chains in chymotrypsin and trypsin, respectively. However, in porcine elastase 1, the corresponding residue is threonine. Consistent with the identified structural alteration, thrombin Quick II incorporates [3H]diisopropyl fluorophosphate stoichiometrically and hydrolyzes the elastase substrate succinyl-Ala-Ala-Pro-Leu-p-nitroanilide with a relative kcat/KM of 0.14 when compared to thrombin. This results from a 3-fold increase in KM and a 2.5-fold decrease in kcat for thrombin Quick II when compared to thrombin acting on the same substrate. These results and those of other investigators studying mutant trypsins support the conclusion that the catalytic activity of serine proteases is very sensitive to structural alterations in the primary substrate binding pocket.
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PMID:Substitution of valine for glycine-558 in the congenital dysthrombin thrombin Quick II alters primary substrate specificity. 271 46

We have isolated and analyzed the gene for batroxobin, a thrombin-like snake venom enzyme. Three overlapping DNA segments containing the entire batroxobin gene were identified. Sequence analysis revealed that the batroxobin gene spans 8 kilobase pairs and contains five exons. Mature batroxobin is encoded by four separate exons, 2 to 5. The catalytic residues of batroxobin, His-41, Asp-86, and Ser-178, are encoded by separate exons, exons 2, 3, and 5, respectively. The exon/intron organization of the batroxobin gene is different from that of the prothrombin gene but very similar to those of the trypsin and kallikrein genes. These results indicate that batroxobin is not a member of the prothrombin family but one of the trypsin/kallikrein family. The snake venom gland is assumed to originate from the submaxillary gland. Therefore, batroxobin is expected to be a member of the glandular kallikrein family.
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PMID:Organization of the gene for batroxobin, a thrombin-like snake venom enzyme. Homology with the trypsin/kallikrein gene family. 316 91

Trypsin, porcine pancreatic kallikrein, and several blood coagulation enzymes, including bovine thrombin, bovine factor Xa, human factor Xa, human plasma factor XIa, human plasma factor XIIa, and human plasma kallikrein, were inactivated by a number of substituted isocoumarins containing basic functional groups (aminoalkoxy, guanidino, and isothiureidoalkoxy). 3-Alkoxy-4-chloro-7-guanidinoisocoumarins were found to be the most potent inhibitors for the coagulation enzymes tested with kobsd/[I] values in the range of 10(3)-10(5) M-1 s-1. 4-Chloro-3-isothiureidoalkoxyisocoumarins show high inhibitory potency toward porcine pancreatic kallikrein, human plasma kallikrein, human factor XIa, human factor XIIa, and trypsin with kobsd/[I] values of the order of 10(4)-10(5) M-1 s-1. The inhibition of these serine proteases by the substituted isocoumarins are time dependent, and the inactivation of trypsin by 3-alkoxy-4-chloro-7-guanidinoisocoumarins and 7-amino-4-chloro-3-(3-isothiureidopropoxy)isocoumarin occured concurrently with the loss of the isocoumarin absorbance. The complex formed from inactivation of trypsin by these two types of inhibitors was very stable and regained less than 4% activity in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (pH 7.5) after 1 day at 25 degrees C and regained 8-45% activity upon addition of buffered 0.29 M hydroxylamine. Trypsin inactivated by other inhibitors regained full activity upon standing or addition of hydroxylamine. Thrombin inactivated by 3-alkoxy-4-chloro-7-guanidinoisocoumarins was also quite stable and only regained 9-15% activity under similar conditions. These results are consistent with a proposed mechanism, where serine proteases inactivated by aminoalkoxyisocoumarins or isothiureidoalkoxyisocoumarins form acyl enzymes that will deacylate upon standing or addition of hydroxylamine. However, the acyl enzymes formed from 3-alkoxy-4-chloro-7-guanidinoisocoumarins or 7-amino-4-chloro-3-(3-isothiureidopropoxy)-isocoumarin will decompose further, probably through a quinone imine methide, to give an irreversibly inactivated enzyme by reaction with an active-site nucleophile such as His-57. The quinone imine methide intermediate may also react with a solvent nucleophile to give an acyl enzyme that can be reactivated by hydroxylamine. The inhibitors 4-chloro-7-guanidino-3-methoxyisocoumarin and 4-chloro-3-ethoxy-7-guanidinoisocoumarin have been tested as anticoagulants in human plasma and were effective at prolonging the prothrombin time. However, they are unstable in plasma (t1/2 = 4-8 min), and their in vivo utility may be limited.
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PMID:Mechanism-based isocoumarin inhibitors for trypsin and blood coagulation serine proteases: new anticoagulants. 316 17

Antithrombin III (AT III) inhibits thrombin via an arginine-serine interaction. Insoluble polystyrene resins grafted with arginyl methyl ester have been synthesized, and their interaction with thrombin tested. One of these resins was selected for its high affinity for thrombin. In this paper we report the characteristics of this thrombin resin interaction. Using this substituted polystyrene resin as a support for affinity chromatography, we have compared the binding of thrombin with that of other proteins (prothrombin, Factor IXa, trypsin and AT III). It was found that 0.7 mg of highly purified human thrombin (2,100 U/mg) was bound to 1 g of resin. This could only be eluted at high ionic strength (1.5 M) and the amidolytic and clotting activities of the eluted thrombin remained unchanged. The binding of thrombin to the resin involves the active site of the enzyme but also other residues since, when DIP thrombin was used, the inactive enzyme could be eluted at lower ionic strength (1.0 M). This resin seems to be specific for thrombin because it does not bind the other serine-proteases (trypsin or Factor IXa), prothrombin (the inactive precursor of thrombin) or AT III. The arginyl residues of the resin are important for the specificity of the interaction with Factor IIa since prolyl residues are totally ineffective. Chromatography performed on such a resin is a very efficient method of purifying thrombin, and may be very useful for the removal of thrombin as a contaminant of plasma protein fractions.
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PMID:Thrombin binding properties of insoluble modified polystyrene: Part II. 326 Jun 95

A low molecular weight serine protease inhibitor, named trypstatin, was purified from rat peritoneal mast cells. It is a single polypeptide with 61 amino acid residues and an Mr of 6610. Trypstatin markedly inhibits blood coagulation factor Xa (Ki = 1.2 x 10(-10) M) and tryptase (Ki = 3.6 x 10(-10) M) from rat mast cells, which have activities that convert prothrombin to thrombin. It also inhibits porcine pancreatic trypsin (Ki = 1.4 x 10(-8) M) and chymase (Ki = 2.4 x 10(-8) M) from rat mast cells, but not papain, alpha-thrombin, or porcine pancreatic elastase. Trypstatin forms a complex in a molar ratio of 1:1 with trypsin and one subunit of tryptase. The complete amino acid sequence of this inhibitor was determined and compared with those of Kunitz-type inhibitors. Trypstatin has a high degree of sequence homology with human and bovine inter-alpha-trypsin inhibitors, A4(751) Alzheimer's disease amyloid protein precursor, and basic pancreatic trypsin inhibitor. However, unlike other known Kunitz-type protease inhibitors, it inhibits factor Xa most strongly.
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PMID:Kunitz-type protease inhibitor found in rat mast cells. Purification, properties, and amino acid sequence. 326 66

The effects of specific low- and high-molecular weight inhibitors of chymase and tryptase and F(ab')2 of antichymase on histamine release from activated mast cells were examined. The release of histamine induced by anti-rat immunoglobulin E was markedly inhibited by F(ab')2 fragments of antichymase and the low-molecular weight inhibitor of chymase chymostatin, whereas release of histamine induced by calcium ionophore A23187 was inhibited only by chymostatin. Neither the inhibitor nor the antibody affected histamine release induced by compound 48/80. These results suggest that two main chymotrypsin-type proteases are involved in process of degranulation: one is chymase, which acts at a step before calcium entry, and the other is an unidentified protease, which acts at a step after calcium entry. These results are summarized in Figure 8. After degranulation, released chymase remains associated with the cell surface while released tryptase was found in the extracellular milieu. Tryptase converted bovine prothrombin to thrombin, as shown by increase in thrombin activity with a synthetic substrate, t-butyloxy-carbonyl-Val-Pro-Arg-4-methyl-coumaryl-7-amide. The apparent Km value toward prothrombin was relatively low (2.3 microM), suggesting that tryptase contributes to blood coagulation or the process of fibrosis in tissues. The proteolytic products of IgG1 produced by chymase had chemotactic activity for neutrophil leukocytes in vitro and in vivo. These findings indicate the possible functions of these proteases after degranulation.
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PMID:Biological functions of serine proteases in the granules of rat mast cells. 354 4

Bacterial protein, staphylocoagulase, binds stoichiometrically to human prothrombin resulting in a coagulant complex, staphylothrombin. The enzymatic properties of staphylothrombin differ from those of alpha-thrombin in their substrate specificities toward natural and synthetic substrates, in addition to their interaction with protease inhibitors. In order to obtain information about the region of staphylocoagulase that interacts with human prothrombin, staphylocoagulase was cleaved by alpha-chymotrypsin. This limited alpha-chymotryptic cleavage of staphylocoagulase yielded three large fragments, fragments of 43, 30, and 20 kDa. The 43-kDa fragment exhibited a high affinity for human prothrombin (Kd = 1.7 nM), which is comparable to the affinity observed using intact staphylocoagulase (Kd = 0.46 nM). A complex of the 43-kDa fragment and prothrombin possessed both clotting and amidase activities essentially identical to those observed in a complex of intact staphylocoagulase and prothrombin. The 30-kDa fragment exhibited weaker affinity for prothrombin (Kd = 120 nM). While a complex of this fragment and prothrombin did not exhibit clotting activity, it nonetheless possessed a weak amidase activity. The 20-kDa fragment was found only to bind to prothrombin. The NH2-terminal sequence analyses of these fragments revealed that the 43-kDa fragment constitutes the NH2-terminal portion of staphylocoagulase, and contains the 30-kDa and 20-kDa fragments. It is therefore concluded that the functional region of staphylocoagulase for binding and activation of prothrombin is localized in the NH2-terminal region of the intact protein. The 43-kDa fragment contains 324 amino acids with a molecular weight of 38,098. The 43-kDa fragment has an unusual amino acid composition based on the sequence, in which the sum of Asp (28 residues), Asn (22), Glu (35), Gln (9), and Lys (52) residues accounts for more than 45% of the total residues. A comparison of the amino acid sequence of the 43-kDa fragment with that of streptokinase did not reveal any obvious sequence homology. There was also no sequence homology with those of trypsin, alpha-chymotrypsin, and elastase.
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PMID:Difference in enzymatic properties between "staphylothrombin" and free alpha-thrombin. 355 30

Prothrombin Barcelona has been isolated from a patient with a normal prothrombin antigen level but low prothrombin coagulant activity. The activation of this protein is impaired by the absence of one of the two factor Xa-catalyzed cleavages that normally lead to the formation of thrombin. Prothrombin Barcelona and prothrombin were isolated from patient plasma and normal plasma, respectively, in a single-step, high-yield immunoaffinity purification using conformation-specific antibodies immobilized on Sepharose. After reduction and alkylation, the purified proteins were subjected to trypsin hydrolysis. The resulting peptides were separated by reverse-phase high performance liquid chromatography. Comparison of the peptide maps of prothrombin Barcelona and prothrombin demonstrated that a peptide, identified as fragment 274-287 in prothrombin by automated Edman degradation, was missing in the prothrombin Barcelona digest. In the chromatogram derived from prothrombin Barcelona, an additional peptide was observed. The amino acid sequence of this peptide was Ala-Ile-Glu-Gly-Cys-Thr-Ala-Thr-Ser-Glu-Tyr-Gln-Thr-Phe-Phe-Asn-Pro-Arg, corresponding to residues 269-287 in prothrombin except for the substitution of cysteine for arginine at residue 273. The substitution of cysteine for arginine was confirmed by tryptic digestion of 14C-carboxymethylated prothrombin Barcelona. Edman degradation of fragment 269-287 indicated the association of 14C with the cysteine at residue 273. The replacement of arginine by cysteine at residue 273, adjacent to the known factor Xa cleavage site, precludes normal activation of prothrombin Barcelona by factor Xa and the generation of thrombin.
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PMID:Molecular defect of prothrombin Barcelona. Substitution of cysteine for arginine at residue 273. 377 62

The effect of tryptase purified from rat peritoneal mast cells on bovine prothrombin was examined. Tryptase activated prothrombin, as evidenced by the increase in thrombin activity with a synthetic substrate, t-butyloxy-carbonyl-Val-Pro-Arg-4-methylcoumaryl-7-amide. The apparent Km value toward bovine prothrombin and the kcat value were 2.3 microM and 46.3 s-1, respectively. Studies on the time course of prothrombin activation by tryptase and by activated factor X (Xa), and analysis of the activation products on sodium dodecyl sulfate gel electrophoresis showed that the process of activation of prothrombin by tryptase was similar to that by Xa except that an intermediate of 67,000 daltons was formed.
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PMID:Tryptase from rat mast cells converts bovine prothrombin to thrombin. 390 53

The primary structure of the procoagulant- and prothrombin-binding domains, the 43- and 30-kDa fragments previously isolated from staphylocoagulase, has been determined by sequencing peptides derived from various chemical (CNBr and 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine) and enzymatic (trypsin and alpha-chymotrypsin) cleavages. Carboxypeptidase Y was also used to deduce the COOH-terminal sequence. The 43-kDa fragment contained 324 amino acids and had a calculated molecular weight of 38,098. It included the entire structure of the 30-kDa fragment located in the COOH-terminal portion (positions 126-324). The 43-kDa fragment had an unusual amino acid composition based on the sequence, in which the sum of Asp (28 residues), Asn (22), Glu (35), Gln (9), and Lys (52) residues accounted for more than 45% of the total. In addition, the frequent occurrence of repetitions of the various kinds of dipeptides was found along the whole sequence. Structural comparison of the NH2-terminal portion of the 43-kDa fragment of staphylocoagulase with that of streptokinase did not reveal any obvious sequence homologies. There was also no sequence homology with that of trypsin, alpha-chymotrypsin, and elastase.
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PMID:The amino acid sequence of the procoagulant- and prothrombin-binding domain isolated from staphylocoagulase. 394 Oct 89


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