Abstract

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, Calpha-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 firstly with respect to their structure (topographically), and secondly with respect to their free energy (thermodynamically). The alanine residue substrate was cleaved best by elastase, the phenylalanine substrate by chymotrypsin. Trypsin cleaved two 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 two to seven carbon atoms. Elastase cleaved tri, tetra, and pentapeptides of alanine. p-Nitroanilides of the glycine series, as well as 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 found to be 1.8 nm in elastolytic substrates.