Abstract Anionic chromogenic substrates, 3-carboxypropionyl tripeptide p-nitroanilides modified with glycine, β-alanine, alanine, leucine and proline in positions P 1, P 2, and P 3 were synthesized. The substrates were digested with pancreatic elastase and the values of K m, k cat, and k cat/K m were determined. Alanine plays a decisive role in position P 1, substrates with glycine or β-alanine in this position are not cleaved. The substitution in P 2 ...