MAPPING THE EXTENDED SUBSTRATE BINDING-SITE OF CATHEPSIN-G AND HUMAN-LEUKOCYTE ELASTASE - STUDIES WITH PEPTIDE-SUBSTRATES RELATED TO THE ALPHA-1-PROTEASE INHIBITOR REACTIVE SITE

The kinetic constants for the hydrolysis of a series of 4-nitroanilide substrates by human leukocyte (HL) elastase and cathepsin G, porcine pancreatic elastase, and bovine chymotrypsin at pH 7.50 are reported. HL elastase and cathepsin G are currently thought to be the agents responsible for destruction of the lung in the disease emphysema. MeO-Suc-Ala-Ala-Pro-Val-NA [MeO-Suc, methoxysuccinyl; NA, 4-nitroanilide] is an excellent substrate for HL elastase and is not hydrolyzed by cathepsin G. The MeO-Suc-group increases the solubility of a substrate relative to the acetyl group. With HL elastase, this structural change increases the reactivity of the enzyme toward both 4-nitroanilide substrates and chloromethyl ketone inhibitors. This indicates that HL elastase is interacting with at least 5 residues of a substrate (or inhibitor). Cathepsin G prefers P5 groups which are negatively charged such as Suc-, Suc(4F)-, Glt-, or Mal- [Suc(4F), perfluorosuccinyl; Glt, glutaryl; Mal, malonyl]. This enzyme, in common with many other serine proteases, cannot accept a Pro residue at its S3 subsite. One of the better substrates for cathepsin G, Suc-Ala-Ala-Pro-Phe-NA, was not hydrolyzed by HL elastase. These tools should be useful in the study of the biological function of HL elastase and cathepsin G. Two tetrapeptide 4-nitroanilide substrates related to the reactive site of the plasma .alpha.1-protease inhibitor (.alpha.1-PI) (.alpha.1-antitrypsin) were studied. Both have a P1 Met residue and one, MeO-Suc-Ala-Ile-Pro-Met-NA, has the exact sequence of the P4-P1 residues at the proteolysis site of .alpha.1-PI. Both MeO-Suc-Ala-Ala-Pro-Met-NA and MeO-Suc-Ala-Ile-Pro-Met-NA react with cathepsin G, HL elastase, and bovine chymotrypsin. The former is the best 4-nitroanilide substrate of cathepsin G yet reported. Oxidation of MeO-Suc-Ala-Ala-Pro-Met-NA yielded 2 diastereomeric sulfoxides. Neither are bound to or was hydrolyzed by HL elastase or cathepsin G. Both reacted poorly with bovine chymotrypsin. Previous work showed that oxidation of .alpha.1-PI destroys its inhibitory activity. Oxidation of the P1 Met of .alpha.1-PI is capable to destroying its reactivity toward most serine proteases. Oxidation of .alpha.1-PI by some component in cigarette smoke would offer 1 explanation in molecular terms for the link between smoking and emphysema.