Purification and characterization of a novel zinc-proteinase from cultures of Aeromonas hydrophila

J Biol Chem. 1993 Apr 25;268(12):9071-8.

Abstract

While searching for an enzyme capable of breaking epsilon-(gamma-Glu)-Lys isopeptide bonds cross-linking protein chains, we purified a metallo-proteinase which mimics the action of an isopeptidase on the gamma-chain dimers of cross-linked fibrin. The enzyme is present in the growth medium of the bacterium Aeromonas hydrophila, isolated from the intestinal 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 synthetic peptides suggest that the enzyme prefers Gly-Gly or acetyl-Gly in the P2 and P1 positions, respectively (Schecter, I., and Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162). Nonpolar amino acid residues seem to be favored in the P1' and P2' positions. The enzyme contains one atom of zinc and is inhibited by 1,10-phenanthroline, but not by EDTA. Iodoacetate, leupeptin, diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, pepstatin, and alpha 2-macroglobulin have no effect on enzyme activity. Disulfide reducing reagents, such as dithiothreitol or 2-mercaptoethanol, 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 with the LasA protein from Pseudomonas aeruginosa.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Aeromonas hydrophila / enzymology*
  • Amino Acid Sequence
  • Chromatography, High Pressure Liquid
  • Electrophoresis, Polyacrylamide Gel
  • Humans
  • Hydrogen-Ion Concentration
  • Metalloendopeptidases / chemistry
  • Metalloendopeptidases / genetics
  • Metalloendopeptidases / isolation & purification*
  • Metalloendopeptidases / metabolism
  • Molecular Sequence Data
  • Sequence Homology, Amino Acid
  • Substrate Specificity

Substances

  • Metalloendopeptidases
  • microbial metalloproteinases