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.
Highlights
Mast cells (MC) are distributed along both external and internal surfaces of the body
Mucosal MC, another subtype of MC, are found in the mucosa of the airways and the intestine. Due to their tissue location MC are among the first cells to encounter bacteria, viruses and other foreign material that enter our tissues [1]. These cells store a large amount of potent mediators in their cytoplasmic granules and the majority of the proteins found within these granules are serine proteases [2,3,4,5]
We have studied the role of these two aa in conferring specificity to the cleavage of angiotensin I (Ang I) to Ang II
Summary
Mast cells (MC) are distributed along both external and internal surfaces of the body. Mucosal MC, another subtype of MC, are found in the mucosa of the airways and the intestine Due to their tissue location MC are among the first cells to encounter bacteria, viruses and other foreign material that enter our tissues [1]. These cells store a large amount of potent mediators in their cytoplasmic granules and the majority of the proteins found within these granules are serine proteases [2,3,4,5] These abundant granule proteases are stored in tight complexes with negatively charged proteoglycans and are released into the extracellular environment in response to immunological and neuronal stimuli [6,7]. The a-chymases are found as a single gene in all species investigated, except for ruminants where two very similar a-chymase genes have been identified [12], whereas functional b-chymases have only been identified in rodents
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have