SUBSTRATE RECOGNITION BY RECOMBINANT SERINE COLLAGENASE-1 FROM UCA PUGILATOR

Uca pugilator serine collagenase 1 was cloned and sequenced from a fid dler crab hepatopancreas cDNA library. A full-length sequence encodes a 270-amino acid pre-pro-enzyme highly identical in structure to the c hymotrypsin 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/glyc eraldehyde-3-phosphate dehydrogenase promoter. Upon activation with tr ypsin, the recombinant collagenase possesses collagenolytic properties identical to those of the enzyme isolated from the crab hepatopancrea s. The collagenase substrate binding pocket recognizes a wide range of basic, hydrophobic, and neutral polar residues. beta-Branched and aci dic amino acids are poor substrates. Acylation is rate-limiting for co llagenase versus peptidyl amides, rather than deacylation, as for tryp sin and chymotrypsin. Correlations relating substrate volume and hydro phobicity to catalysis were found for collagenase and compared to thos e for chymotrypsin and elastase. Relative enzyme efficiencies on singl e amino acid versus tetrapeptide amide substrates show that collagenas e derives less catalytic efficiency from binding of the primary substr ate residue than trypsin or chymotrypsin, but compensates in binding o f the extended peptidyl residues. Serine collagenase 1 is a novel memb er of the chymotrypsin protease family, by virtue of its amino acid se quence and multifunctional active site.