Abstract
Chymotrypsin-like serine proteases are found in high abundance in mast cell granules. By site-directed mutatgenesis, we have previously shown that basic amino acids in positions 143 and 192 (Arg and Lys respectively) of the human mast cell chymase are responsible for an acidic amino acid residue preference in the P2' position of substrates. In order to study the influence of these two residues in determining the specificity of chymase inhibitors, we have synthesized five different potent inhibitors of the human chymase. The inhibitory effects of these compounds were tested against the wild-type enzyme, against two single mutants Arg143Gln and Lys192Met and against a double mutant, Arg143Gln+Lys192Met. We observed a markedly reduced activity of all five inhibitors with the double mutant, indicating that these two basic residues are involved in conferring the specificity of these inhibitors. The single mutants showed an intermediate phenotype, with the strongest effect on the inhibitor by the mutation in Lys192. The Lys192 and the double mutations also affected the rate of cleavage of angiotensin I but did not seem to affect the specificity in the cleavage of the Tyr4-Ile5 bond. A more detailed knowledge about which amino acids that confer the specificity of an enzyme can prove to be of major importance for development of highly specific inhibitors for the human chymase and other medically important enzymes.