Mapping of the substrate-binding site of the human granulocyte elastase by the aid of tripeptidyl-p-nitroanilide substrates

The kinetic properties of the human granulocyte elastase /EC 3.4.21.11/ were investigated with 24 tripeptidyl-pNA substrates. By the regression analysis of the kinetic data obtained with 15 substrates a relatively hydrophobic compound, Boc-D-Phe-Ala-Nle-pNA, was predicted as the optimal substrate sequence. The compound was synthesized, assayed and the predicted K_m = 4.2 uM was confirmed experimentally. The substrate-binding site of granulocyte elastase appeared to be hydrophobic and very much similar to that of the pancreatic enzyme at the S₂–S₄ subsites, but the S₁ subsite, which determines the primary specificity, could accomodate bulkier residues and it was less selective than that in the pancreatic enzyme.