Mercurial activation of human PMN leucocyte type IV procollagenase (gelatinase)

Abstract

Autoproteolytic activation and processing of human polymorphonuclear leucocyte (PMNL) type IV procollagenase (gelatinase) was initiated by HgCl 2 and was investigated by kinetic analysis and N-terminal sequence determination of the reaction products. In the first instance the propeptide domain was lost by subsequent cleavage of the Asp 15-Leu 16, Glu 40-Met 41, Leu 52-Leu 53 and Ala 74-Met 75 peptide bonds. The PRCGVPD sequence motif (residues Pro 78-Asp 84), which is conserved in all metalloproteinases and expected to be relevant for latency, remained uncleaved at the activated enzyme. The generated intermediate was further processed by three C-terminal cleavages. The Glu 666-Leu 667, Ala 506-Glu 507 and Ala 398-Leu 399 bonds were hydrolysed sucessively. From the fragmentation products we were able to conclude that three released fragment peptides contained unpaired free cysteine with the residues Cys 497, Cys 653, Cys 683. Cleavage of the first C-terminal peptide bond resulted in the loss of one of the conserved Cys residues of the hemopexin-like domain, whereas the Cys residue of the PRCGVPD motif was retained at the fully active enzyme. The possibility of an entirely different activation mechanism for PMNL type IV procollagenase is discussed.