PURIFICATION AND CHARACTERIZATION OF A NOVEL ZINC-PROTEINASE FROM CULTURES OF AEROMONAS-HYDROPHILA

While searching for an enzyme capable of breaking epsilon-(gamma-Glu)- Lys isopeptide bonds cross-linking protein chains, we purified a metal lo-proteinase which mimics the action of an isopeptidase on the gamma- chain dimers of cross-linked fibrin. The enzyme is present in the grow th medium of the bacterium Aeromonas hydrophila, isolated from the int estinal tract of the leech Hirudo medicinalis. It is a 19-kDa protein which specifically hydrolyzes the Gly-Ala peptide bond within the Gly- Gly-Ala sequence, located near the cross-link site in the gamma-chain dimer of fibrin. Substrate specificity studies with a number of synthe tic peptides suggest that the enzyme prefers Gly-Gly or acetyl-Gly in the P2 and P1 positions, respectively (Schecter, I., and Berger, A. (1 967) Biochem. Biophys. Res. Commun. 27, 157-162). Nonpolar amino acid residues seem to be favored in the P1' and P2' positions. The enzyme c ontains one atom of zinc and is inhibited by 1,10-phenanthroline, but not by EDTA. Iodoacetate, leupeptin, diisopropyl fluorophosphate, phen ylmethylsulfonyl fluoride, pepstatin, and alpha2-macroglobulin have no effect on enzyme activity. Disulfide reducing reagents, such as dithi othreitol or 2-mereaptoethanol, inactivate the enzyme completely. The partial amino-terminal sequence shows 46% identity with a zinc metallo -proteinase from a strain of Lysobacter enzymogenes and 69% identity w ith the LasA protein from Pseudomonas aeruginosa.