Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.9 (enterokinase)
 675 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To test the role of Asp-189 which is located at the base of the substrate binding pocket in determining the specificity of trypsin toward basic substrates, this residue was replaced with a lysine residue by site-directed mutagenesis. Both rat trypsinogen and Lys-189 trypsinogen were expressed and secreted into the periplasmic space of Escherichia coli. The proteins were purified to homogeneity and activated by porcine enterokinase, and their catalytic activities were determined on natural and synthetic substrates. Lys-189 trypsin displayed no catalytic activity toward arginyl and lysyl substrates. Further, there was no compensatory change in specificity toward acidic substrates; no cleavage of aspartyl or glutamyl bonds was detected. Additional studies of substrate specificity involving gas-phase sequence analyses of digested natural substrates revealed an inherent but low chymotrypsin-like activity of trypsin. This activity was retained but modified by the Asp to Lys change at position 189. In addition to hydrolyzing phenylalanyl and tyrosyl peptide bonds, the mutant enzyme has the unique property of cleaving leucyl bonds. On the basis of computer graphic modeling studies of the Lys-189 side chain, it appears that the positively charged NH2 group is directed outside the substrate binding pocket. The resulting hydrophobic cavity may explain the altered substrate specificity of the mutant enzyme. The relatively low chymotrypsin-like activity of both recombinant enzymes may be due to distorted positioning of the scissile bond with respect to the catalytic triad rather than to the lack of sufficient interaction between the hydrophobic side chains and the substrate binding pocket of the enzyme.
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PMID:Selective alteration of substrate specificity by replacement of aspartic acid-189 with lysine in the binding pocket of trypsin. 311 31

Duodenase, a serine protease from bovine duodenum mucosa, was located in endoplasmic reticulum, the Golgi secretory granules of epithelial cells and ducts of Brunner's glands by the A-gold immunocytochemical method. Duodenase exhibits trypsin-like and chymotrypsin-like specificities with a preference for substrates having lysine at the P1 and proline at the P2 positions. The kinetic constants for the hydrolysis of 21 potential duodenase substrates are reported. The best substrates were found to be alpha-N-tosylglycylprolyllysine 4-nitroanilide (k[cat]/Km of 35000 M[-1] s[-1]), alpha-N-succinylthreonylprolyllysine 4-nitroanilide (k[cat]/Km of 18000 M[-1] s[-1]) and alpha-N-serylprolyllysine 4-nitroanilide (k[cat]/Km of 2600 m[-1] s[-1]), all of which contain the P1-P3 sequence of the enteropeptidase zymogen/activation site. On the basis of its catalytic properties and sites of localization, duodenase has been postulated to be an activator of the enteropeptidase precursor. A tetradecapeptide (LVTQEVSPKIVGGS) having the P9-P5'sequence of the cleavage site of zymogen activation of bovine proenteropeptidase was synthesized, and kinetic parameters of its hydrolysis by duodenase were determined (Km of 87 microM; k[cat] of 1.4 s[-1]; k[cat]/Km of 16000 M[-1] s[-1]). Crystals of duodenase frozen in a stream of liquid nitrogen diffracted synchrotron X-rays to 0.2-nm resolution.
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PMID:Subcellular localization, substrate specificity and crystallization of duodenase, a potential activator of enteropeptidase. 937 Mar 74

Activated mast cells release a variety of potent inflammatory mediators including histamine, cytokines, proteoglycans, and serine proteases. The serine proteases belong to either the chymase (chymotrypsin-like substrate specificity) or tryptase (trypsin-like specificity) family. In this report we have investigated the substrate specificity of a recently identified mast cell protease, rat mast cell protease-4 (rMCP-4). Based on structural homology, rMCP-4 is predicted to belong to the chymase family, although rMCP-4 has previously not been characterized at the protein level. rMCP-4 was expressed with an N-terminal His tag followed by an enterokinase site substituting for the native activation peptide. The enterokinase-cleaved fusion protein was labeled by diisopropyl fluorophosphate, demonstrating that it is an active serine protease. Moreover, rMCP-4 hydrolyzed MeO-Suc-Arg-Ala-Tyr-pNA, thus verifying that this protease belongs to the chymase family. rMCP-4 bound to heparin, and the enzymatic activity toward MeO-Suc-Arg-Ala-Tyr-pNA was strongly enhanced in the presence of heparin. Detailed analysis of the substrate specificity was performed using peptide phage display technique. After six rounds of amplification a consensus sequence, Leu-Val-Trp-Phe-Arg-Gly, was obtained. The corresponding peptide was synthesized, and rMCP-4 was shown to cleave only the Phe-Arg bond in this peptide. This demonstrates that rMCP-4 displays a striking preference for bulky/aromatic amino acid residues in both the P1 and P2 positions.
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PMID:Rat mast cell protease 4 is a beta-chymase with unusually stringent substrate recognition profile. 1189 50

Chymases are mast cell serine proteases with chymotrypsin-like primary substrate specificity. Amino acid sequence comparisons of alpha-chymases from different species indicated that certain rodent alpha-chymases have a restricted S1 pocket that could only accommodate small amino acids, i.e. they may, despite being classified as chymases, in fact display elastase-like substrate specificity. To explore this possibility, the alpha-chymase, rat mast cell protease 5 (rMCP-5), was produced as a proenzyme with a His6 purification tag and an enterokinase-susceptible peptide replacing the natural propeptide. After removal of the purification tag/enterokinase site by enterokinase digestion, rMCP-5 bound the serine-protease-specific inhibitor diisopropyl fluorophosphate, showing that rMCP-5 was catalytically active. The primary specificity was investigated with chromogenic substrates of the general sequence succinyl-Ala-Ala-Pro-X-p-nitroanilide, where the X was Ile, Val, Ala, Phe or Leu. The activity was highest toward substrates with Val or Ala in the P1 position, whereas low activity toward the peptide with a P1 Phe was observed, indicating that the substrate specificity of rMCP-5 indeed is elastase-like. The extended substrate specificity was examined utilizing a phage-displayed random nonapeptide library. The preferred cleavage sequence was resolved as P4-(Gly/Pro/Val), P3-(Leu/Val/Glu), P2-(Leu/Val/Thr), P1-(Val/Ala/Ile), P1'-(Xaa), and P2'-(Glu/Leu/Asp). Hence, the extended substrate specificity is similar to human chymase in most positions except for the P1 position. We conclude that the rat alpha-chymase has converted to elastase-like substrate specificity, perhaps associated with an adoption of new biological targets, separate from those of human alpha-chymase.
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PMID:Extended substrate specificity of rat mast cell protease 5, a rodent alpha-chymase with elastase-like primary specificity. 1290 Apr 23

Corin is a cardiac transmembrane serine protease. In cell-based studies, corin converted pro-atrial natriuretic peptide (pro-ANP) to mature ANP, suggesting that corin is potentially the pro-ANP convertase. In this study, we evaluated the importance of the transmembrane domain and activation cleavage in human corin. We showed that a soluble corin that consists of only the extracellular domain was capable of processing recombinant human pro-ANP in cell-based assays. In contrast, a mutation at the conserved activation cleavage site, R801A, abolished the function of corin, demonstrating that the activation cleavage is essential for corin activity. These results allowed us to design, express, and purify a mutant soluble corin, EKsolCorin, that contains an enterokinase recognition sequence at the activation cleavage site. Purified EKsolCorin was activated by enterokinase in a dose-dependent manner. Activated EK-solCorin had hydrolytic activity toward peptide substrates with a preference for Arg and Lys residues in the P-1 position. This activity of EKsolCorin was inhibited by trypsin-like serine protease inhibitors but not inhibitors of chymotrypsin-like, cysteine-, or metallo-proteases. In pro-ANP processing assays, purified active EKsolCorin converted recombinant human pro-ANP to biologically active ANP in a highly sequence-specific manner. The pro-ANP processing activity of EKsolCorin was not inhibited by human plasma. Together, our data indicate that the transmembrane domain is not necessary for the biological activity of corin but may be a mechanism to localize corin at specific sites, whereas the proteolytic cleavage at the activation site is an essential step in controlling the activity of corin.
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PMID:Functional analysis of the transmembrane domain and activation cleavage of human corin: design and characterization of a soluble corin. 1455 95

The synthetic gene encoding human enteropeptidase light chain (L-HEP) was cloned into plasmid pET-32a downstream from the gene of fusion partner thioredoxin immediately after the DNA sequence encoding the enteropeptidase recognition site. The fusion protein thioredoxin (Trx)/L-HEP was expressed in Escherichia coli BL21(DE3). Autocatalytic cleavage of the fusion protein and activation of recombinant L-HEP were achieved by solubilization of inclusion bodies and refolding of Trx/L-HEP fusion protein. The kinetic parameters of human and bovine enteropeptidases in the presence of different concentrations of Ca2+ and Na+ for cleavage of the specific substrate GD4K-na and nonspecific substrates such as small ester Z-Lys-SBzl and chromogenic substrates Z-Ala-X-Arg-pNA have been comparatively analyzed. It is demonstrated that positively charged ions increased the Michaelis constant (Km) for cleavage of specific substrate GD4K-na, while the catalytic constant (k(cat)) remained practically unchanged. L-HEP demonstrated secondary specificity to the chromogenic substrate Z-Ala-Phe-Arg-pNA with k(cat)/Km 260 mM(-1) x sec(-1). Enzymatic activity of L-HEP was suppressed by inhibitors of trypsin-like and cysteine (E-64), but not metallo-, amino-, or chymotrypsin-like proteinases. L-HEP was active over a broad range of pH (6-9) with optimum activity at pH 7.5, and it demonstrated high stability to different denaturing agents.
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PMID:Biochemical characterization of human enteropeptidase light chain. 1648 14

Members of the trypsin-like and chymotrypsin-like kallikrein family are important in the desquamation process. In this study, we isolated cDNA clones encoding trypsinogen 4 (brain trypsinogen) and a previously unreported isoform of trypsinogen from a human keratinocyte cDNA library. The nucleotide sequence of the new isoform only differs from those of trypsinogen 3 (mesotrypsinogen) and trypsinogen 4 in an exon encoding the N-terminal region, indicating that this isoform is an alternative splicing variant of the mesotrypsinogen gene PRSS3. Both isoforms contained the sequence DDDDK-I, a putative cleavage site for activation by enteropeptidase. Thus, after activation, mesotrypsin would be produced. Immunohistochemical and in situ hybridization studies revealed that trypsinogens were expressed and localized in the upper epidermis, especially in the granular layer. In cultured keratinocytes, enteropeptidase mRNA was expressed at the confluent stage, and its expression was strongly upregulated after air exposure. Interestingly, it was synthesized and localized only at the granular layer, suggesting that the generation of active mesotrypsin is restricted to this layer. The enteropeptidase-cleavage product was also found at the same layer. When a skin equivalent model was cultured in the medium without air exposure, the cornified layer was not formed, and many cells expressed trypsinogens and enteropeptidase. Those cells were found to be TUNEL positive. Because mesotrypsin is resistant to naturally occurring trypsin inhibitors, confined expression of the isoforms of mesotrypsinogens and enteropeptidase may indicate that mesotrypsin is involved in keratinocyte terminal differentiation.
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PMID:Keratinocytes synthesize enteropeptidase and multiple forms of trypsinogen during terminal differentiation. 1992 34