Substrate recognition by recombinant serine collagenase 1 from Uca pugilator.
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.
Tsu
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Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0446, USA.
Craik
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D000595:Amino Acid Sequence; D000818:Animals; D060829:Arthropod Proteins; D001483:Base Sequence; D003386:Brachyura; D002417:Cattle; D002918:Chymotrypsin / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D017364:Collagenases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D004064:Digestive System / Q000201:enzymology; D004792:Enzyme Precursors / Q000737:chemistry; D015723:Gene Library; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D017386:Sequence Homology, Amino Acid; D057057:Serine Proteases; D013379:Substrate Specificity; D013552:Swine
D060829:Arthropod Proteins; D017931:DNA Primers; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D057057:Serine Proteases; D002918:Chymotrypsin; C099416:Uca pugilator serine collagenase 1; D017364:Collagenases