Human leukocyte cathepsin G. Subsite mapping with 4-nitroanilides, chemical modification, and effect of possible cofactors

Abstract

The extended substrate binding site of cathepsin G from human leukocytes has been mapped by using a series of peptide 4-nitroanilide substrates. The enzyme has a significant preference for substrates with a P1 Phe over those with the other aromatic amino acids Tyr and Trp. The S2 subsite was mapped with the substrates Suc-Phe-AA-Phe-NA where AA was 13 of the 20 amino acid residues commonly found in proteins. The best residues were Pro and Met. The S3 subsite was mapped with the sequence Suc-AA-Pro-Phe-NA by using 14 different amino acid residues for AA. The two best residues were the isosteric Val and Thr. No significant improvement in reactivity was obtained by extending the substrate to include seven different P4 residues. The kinetic parameters for cathepsin G are significantly slower than those for many other serine proteases. Changes in the reaction conditions and addition of possible cofactors or ligands were in general found to have little effect on the enzymatic activity, while chemical modifications and proteolysis destroyed the activity of cathepsin G. Cathepsin G hydrolyzed peptides containing model desmosine residues and prefers the hydrophobic picolinoyllysine derivative over lysine by substantial margins at both the S4 and S2 subsites but will not tolerate it at S3. Substrates with sequences related to the cathepsin G cleavage site in angiotensin I and angiotensinogen, and the reactive site of alpha 1-antichymotrypsin, were hydrolyzed effectively by enzyme, but with unexceptional rates. Our results indicate that the natural substrate(s) and function(s) of cathepsin G still remain to be discovered.

Enzymatic degradation of endothelin-1 by activated human polymorphonuclear neutrophils

“…Incubation of unlabelled ET-1 with purified cathepsin G allowed to identify a major cleavage site corresponding to the Hisl6-Leu x7 bond, leading to the formation of inactive [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] fragments and the C-terminal pentapeptide.…”

“…This specificity is observed in the hydrolysis of the His-Leu bond of ET-1. The cleavage of this bond, generating [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] fragments and the C-terminal pentapeptide (Table I) is sufficient to induce a biological activity loss of ET-1. Indeed, we have previously shown that [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] peptides, resulting from the hydrolysis of ET-1 by endopeptidase 24.11, were devoid of vasoconstrictor effect on isolated rat aortic rings [9].…”

“…Cathepsin G concentration was determined by measuring free p-nitroanilide released from the substrate N-Suc-Ala-Ala-Pro-Phe-p-nitroanilide [14].…”

“…5). They were collected and submitted to amino acid analysis, Peaks 1, 2, 3 had amino acid compositions compatible with the sequence ET-1 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] and probably differed from each other by additional cleavages in the disulfidelinked loops (Table I) had a composition identical to that of ET-1 and might differ from the intact substrate by one or more cleavages inside the disulfide-linked loops. Peak 6 was present as a double peak due to the appearance of a metabolite not resolved from ET-1.…”

Generation of Gla‐domainless FVIIa by cathepsin G‐mediated cleavage

“…The enzyme cleaves predominantly after Phe, Ala or Leu, with a substantial prel'erence for Phe over the other two aromatic amino acids, Tyr and Trp [11].…”