Abstract
Uca pugilator serine collagenase 1 was cloned and sequenced from a fiddler crab hepatopancreas cDNA library. A full-length sequence encodes a 270-amino acid pre-pro-enzyme highly identical in structure to the chymotrypsin family of serine proteases. The zymogen form of the enzyme was expressed in Saccharomyces cerevisiae as a fusion with the alpha-factor signal sequence under control of the alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase promoter. Upon activation with trypsin, the recombinant collagenase possesses collagenolytic properties identical to those of the enzyme isolated from the crab hepatopancreas. The collagenase substrate binding pocket recognizes a wide range of basic, hydrophobic, and neutral polar residues. beta-Branched and acidic amino acids are poor substrates. Acylation is rate-limiting for collagenase versus peptidyl amides, rather than deacylation, as for trypsin and chymotrypsin. Correlations relating substrate volume and hydrophobicity to catalysis were found for collagenase and compared to those for chymotrypsin and elastase. Relative enzyme efficiencies on single amino acid versus tetrapeptide amide substrates show that collagenase derives less catalytic efficiency from binding of the primary substrate residue than trypsin or chymotrypsin, but compensates in binding of the extended peptidyl residues. Serine collagenase 1 is a novel member of the chymotrypsin protease family, by virtue of its amino acid sequence and multifunctional active site.
Highlights
The chymotrypsin family of serine proteases is a paradigm for enzymic substrate recognition
We present here the cloning, expression, and characterization of crab serine collagenase 1
These criteria show serine collagenase 1 to be a novel member of the chymotrypsin protease family
Summary
RNA Isolation and cDNA Library Construction—Live fiddler crabs (U. pugilator) were obtained from Gulf Specimen Marine Laboratory (Panacea, FL). Action (PCR) was used to amplify a fragment of the crab collagenase cDNA from the U. pugilator hepatopancreas library. Excision and rescue of the Bluescript plasmid containing the cDNA insert was carried out according to the manufacturer’s instructions (Stratagene) Both strands of the cDNA clones comprising the composite map were sequenced by the dideoxy method using Sequenase. Under the conditions of limiting dCTP and high template concentration, the reaction products resembled those of primer extension rather than fragment amplification These homologous probes were hybridized overnight at 50 °C [18]. The activation assay contained 20 l of sample, 5 l of 1 M TPCK-treated bovine trypsin (Sigma), and 200 l of 400 M Suc-AAP-Leu-pNA in 50 mM Tris, 100 mM NaCl, 20 mM CaCl2, pH 8.0. Kinetic Analysis of Recombinant Collagenase, Trypsin, Chymotrypsin, and Elastase—Collagenase was prepared from crab hepatopancreas as described [14]. Kinetic parameters were plotted versus P1 residue volume [26] and the hydrophobicity constant, [27]
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