P-NITROANILIDES OF 3-CARBOXYPROPIONYL-PEPTIDES - THEIR CLEAVAGE BY ELASTASE, TRYPSIN, AND CHYMOTRYPSIN

Fourteen 3-carboxypropionyl-tripeptide-p-nitroanilides of the general formula 3-carboxypropionyl-alanyl-alanyl-Y-p-nitroanilide (Y = glycine, norvaline, S-methylcysteine, valine, norleucine, S-ethylcysteine, methionine, leucine, isoleucine, phenylalanine, tyrosine, S-benzylcysteine, C.alpha.-phenylglycine and proline) were synthesized and their cleavage by elastase, trypsin and chymotrypsin (Km, kcat and kcat/Km) was determined. The significance of amino acid residues in the position of Y was evaluated with respect to their structure (topographically), and free energy (thermodynamically). The alanine residue substrate was cleaved best by elastase, the phenylalanine substrate by chymotrypsin. Trypsin cleaved 2 substrates only, that is those containing a phenylalanine and a tyrosine residue. The optimum length of the elastolytic substrates was studied in a series of N-3-carboxypropionyl-(Ala)n-p-nitroanilides (n = 1, 2, 3, 4, 5), N-3-carboxypropionyl-(Gly)n-p-nitroanilides (n = 1, 2, 3), and in p-nitroanilides of fatty acids with 2-7 C atoms. Elastase cleaved tri, tetra, and pentapeptides of alanine. p-Nitroanilides of the glycine series, and p-nitroanilides of fatty acids were not cleaved. 3-Carboxypropionyl-tetra-alanine-p-nitroanilide was the most suitable substrate so far found for elastase cleavage; it is not cleaved by trypsin nor chymotrypsin. The optimum distance between Y and the terminal anionic carboxyl residue was 1.8 nm in elastolytic substrates.