Substrate specificities and inhibition of two hemorrhagic zinc proteases Ht‐c and Ht‐d from Crotalus atrox venom

The proteolytic specificities of two zinc hemorrhagic toxins (Ht‐c and Ht‐d), isolated from Crotalus atrox venom, were investigated by using the oxidized B chain of bovine insulin and synthetic peptide substrates. The enzymes cleaved the Ala 14 ‐Leu 15 bond of the insulin B chain most rapidly and the Tyr 16 ‐Leu 17 slightly more slowly. The His 5 ‐Leu 6 , His 10 ‐Leu 11 , and Gly 23 ‐Phe 24 bonds were also cleaved but at condiserably slower rates. In order to assess the substrate length preferences of the enzymes, peptide analogs of the B chain about the Ala 14 ‐Leu 15 bond were synthesized ranging in length from four to seven residues. The heptapeptide NH 2 ‐Leu‐Val‐Glu‐Ala‐Leu‐Tyr‐Leu‐COOH was the best peptide substrate tested with the other peptides having decreasing k cat / K m values with decreasing length. The tetrapeptide NH 2 ‐Ala‐Leu‐Tyr‐Leu‐COOH was not cleaved by the enzymes. Furthermore, this peptide was shown to serve as a competitive inhibitor of the toxins. The N ‐acetylated pentapeptides and hexapeptides, synthesized to probe the active site environment of the enzymes, were significantly better substrates than their unacetylated counterparts. The toxins had the highest k cat / K m values for the acetylated peptide Ac‐Val‐Ala‐Leu‐Leu‐Ala‐COOH. The data suggest that the toxins may indeed have extended substrate‐binding sites, which may accomodate at least six amino acid residues. The best substrate examined thus far for the toxins is the fluorogenic peptide analog 2‐aminobenzoyl‐Ala‐Gly‐Leu‐Ala‐4‐nitrobenzylamide, suggestive of similarities between the toxins and mammalian collagenases as well as thermolysin. Mechanisms for inhibition of the enzymes were investigated using amino acid hydroxamates, chloromethyl esters, phosphoramidon and the peptide NH 2 ‐Ala‐Leu‐Tyr‐Leu‐COOH. All of these inhibitors had K i values in the 10 −4 M range.