Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.3 (collagenase)
 18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Affinity-based purification and characterization of the collagenolytic serine protease 1 from Uca pugilator (fiddler crab) hepatopancreas shows that the enzyme cleaves the native bovine alpha 1(I) collagen chain carboxyl-terminal to Gln and Arg residues adjacent to the metallocollagenase site. Cleavage carboxyl-terminal to Leu residues is observed in the alpha 2(I) chain and at a secondary site in alpha 1(I). These sites correlate with the preferences observed toward p-nitroanilide substrates varying at the P1 position, for which the specificity (kcat/Km) is Arg > Leu, Phe, Lys > Gln > Ala. Furthermore, collagen cleavage after Gln was found exclusively between two Gln-Arg bonds. The P'1-P'3 specificity of collagenase, as determined by nucleophile acyl transfer, indicated a strong preference for Arg in the P'1 position. Crab collagenase cleaves peptide bonds adjacent to Leu and Gln at the P1 position more efficiently than trypsin, chymotrypsin, or elastase. Moreover, the efficiency of collagenase toward P1-Arg substrates is equivalent to that of trypsin. Crystals of crab collagenase have been grown complexed with the protein inhibitor ecotin. These crystals diffract to better than 2.8 A resolution and belong to the space group P3(2)21 with unit cell dimensions of a = b = 89.0 A, c = 291.7 A.
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PMID:The substrate specificity of Uca pugilator collagenolytic serine protease 1 correlates with the bovine type I collagen cleavage sites. 803 25

Crab collagenolytic serine protease 1 efficiently cleaves peptide bonds directly C-terminal to basic, polar, and hydrophobic amino acids. The crystal structure of this enzyme complexed to the protein inhibitor ecotin at 2.5 A resolution reveals a large primary binding pocket punctuated on one wall by the side chain of aspartate-226. Removal or relocation of this negatively charged group by site-directed mutagenesis generates variant enzymes which retain very high activities toward selected substrates. Full retention of activity toward hydrophobic substrates in collagenase D226G is accompanied by a 10-100-fold reduction in k(cat)/Km toward basic residues. In contrast, restoration of the negative charge in a trypsin-like position in collagenase D226G/G189D regenerates nearly full activity toward basic substrates while introducing a 5-fold decrease in k(cat)/Km toward hydrophobic amino acids. These results imply that the collagenase S1 pocket has multiple distinct binding sites for different amino acid side chains, a suggestion supported by molecular modeling studies based on the crystal structure. The ease of specificity modification in the primary binding site of this serine protease parallels similar observations with the bacterial enzymes alpha-lytic protease and subtilisin, and stands in sharp distinction to the extensive mutagenesis required to alter specificity in trypsin.
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PMID:Structural basis for the broad substrate specificity of fiddler crab collagenolytic serine protease 1. 915 21