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The extended substrate binding sites of several chymotrypsin-like serine proteases, including rat mast cell proteases I and II (RMCP I and II, respectively) and human and dog skin chymases, have been investigated by using peptide 4-nitroanilide substrates. In general, these enzymes preferred a P1 Phe residue and hydrophobic amino acid residues in P2 and P3. A P2 Pro residue was also found to be quite acceptable. The S4 subsites of these enzymes are less restrictive than the other subsites investigated. The substrate specificity of these enzymes was also investigated by using substrates which contain model desmosine residues and peptides with amino acid sequences of the physiologically important substrates angiotensin I and angiotensinogen and alpha 1-antichymotrypsin, the major plasma inhibitor for chymotrypsin-like enzymes. These substrates were less reactive than the most reactive tripeptide reported here, Suc-Val-Pro-Phe-NA. The thiobenzyl ester Suc-Val-Pro-Phe-SBzl was found to be an extremely reactive substrate for the enzymes tested and was 6-171-fold more reactive than the 4-nitroanilide substrate. The four chymotrypsin-like enzymes were inhibited by chymostatin and N-substituted saccharin derivatives which had KI values in the micromolar range. In addition, several potent peptide chloromethyl ketone and substituted benzenesulfonyl fluoride irreversible inhibitors for these enzymes were discovered. The most potent sulfonyl fluoride inhibitor for RMCP I, RMCP II, and human skin chymase, 2-(Z-NHCH2CONH)C6H4SO2F, had kobsd/[I] values of 2500, 270, and 1800 M-1 s-1, respectively. The substrates and inhibitors reported here should be extremely useful in elucidating the physiological roles of these proteases.

Skin is a rich source of proteolytic enzymes (1) and the release of these proteinases may produce serious structural damage. Incubation of skin with commercial preparations of pancreatic trypsin and elastase, and bacterial collagenase, produced separation of the epidermis from the dermis (2-6). Indeed, a solution of 0.25% bovine pancreatic trypsin is routinely used to separate viable epidermis from dermis for tissue culture purposes. Skin does not encounter active pancreatic proteinases in vivo, but there are several neutral cellular endoproteinases that can be found in the skin at reasonable concentrations which could act upon the tissue. One of these is a chymotrypsin-like proteinase isolated from human dermis where it appears to be a mast cell constituent (7-8). Two other neutral serine proteinases, cathepsin G and elastase, located in the cytoplasmic granules of human polymorphonuclear leukocytes, are also of pathophysiological interest (9-12), since polymorphonuclear leukocytes may migrate into the skin in response to inflammatory signals. Cathepsin G exhibits chymotrypsin-like substrate specificity, but it is distinct from the human skin chymotrypsin-like proteinase (8).This study investigates the effect of human skin chymotrypsin-like proteinase, cathepsin G, and elastase on the integrity of whole skin. As will be shown, the primary action of all three proteinases is at the epidermal-dermal junction. Structural alterations in this region were examined by electron microscopy while the degradation of specific proteins was determined by immunohistochemistry using antibodies to several different basement membrane components. The concentrations of proteinase used for our studies were in the range of 10-700 nM; these enzyme concentrations were selected to approach those that might be attained under physiologic or pathologic conditions in skin. They are 100-1,000-fold lower than proteinase concentrations used in previous studies. The proteinases used for these studies are highly purified. Also presented is a procedure for the simultaneous purification of cathepsin G and elastase from the same human leukocyte preparation.