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 I-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-Sue-Ala-Ala-Pro-VaI-NA is an excellent substrate for HL elastase and is not hydrolyzed by cathepsin G. The MeO-Sue-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 I-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 Pb groups which are negatively charged such as Sue-, Suc(4F)-, Glt-, or Mal-. This enzyme, in common with many other serine proteases cannot accept a Pro residue at its Ss subsite. One of the better substrates for cathepsin G, Sue-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 (~1’ protease inhibitor (ai-antitrypsin) were studied. Both have a P1 Met residue and one, MeO-Suc-Ala-Ile-ProMet-NA, has the exact sequence of the Pa to Pt residues at the proteolysis site of al-PI (Johnson, D. A., and Travis, J. (1978) J. BioL Clrem. 253, 7142-7144). Both MeO-Sue-Ala-Ala-Pro-Met-NA and MeO-Suc-Ala-IlePro-Met-NA react with cathepsin G, HL elastase, and bovine chymotrypsin. The former is in fact the best 4nitroanilide substrate of cathepsin G yet reported. Oxidation of MeO-Sue-Ala-Ala-Pro-Met-NA yielded two diastereomeric sulfoxides. Neither are bound to or was hydrolyzed by HL elastase or cathepsin G. Both reacted poorly with bovine chymotrypsin. In the preceding paper, Johnson and Travis (Johnson, D., and Travis, J. (1979) J. BioL Chem. 254, 4022-4026) show that oxidation of al-PI destroys its inhibitory activity. In concert, our results indicate that oxidation of the PI Met of (YePI is capable to destroying its reactivity toward most serine proteases. Oxidation of al-PI by some component in cigarette smoke would offer one explanation in mo-