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  • Title: Substrate specificities and inhibition of two hemorrhagic zinc proteases Ht-c and Ht-d from Crotalus atrox venom.
    Author: Fox JW, Campbell R, Beggerly L, Bjarnason JB.
    Journal: Eur J Biochem; 1986 Apr 01; 156(1):65-72. PubMed ID: 3514216.
    Abstract:
    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 Ala14-Leu15 bond of the insulin B chain most rapidly and the Tyr16-Leu17 slightly more slowly. The His5-Leu6, His10-Leu11, and Gly23-Phe24 bonds were also cleaved but at considerably slower rates. In order to assess the substrate length preferences of the enzymes, peptide analogs of the B chain about the Ala14-Leu15 bond were synthesized ranging in length from four to seven residues. The heptapeptide NH2-Leu-Val-Glu-Ala-Leu-Tyr-Leu-COOH was the best peptide substrate tested with the other peptides having decreasing kcat/Km values with decreasing length. The tetrapeptide NH2-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 kcat/Km 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 accommodate 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 NH2-Ala-Leu-Tyr-Leu-COOH. All of these inhibitors had Ki values in the 10(-4) M range.
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