Résumé
A series of tetrapeptide
p-nitroanilide substrates of the general formula: suc-Ala-Ala-Pro-Aaa-
p-nitroanilide was used to map the S
1 binding pocket of human cathepsin G. Based on the
k
cat/
K
m parameter, the following order of preference was found: Lys=Phe>Arg=Leu>Met>Nle=Nva>Ala>Asp. Thus, the enzyme exhibits clear dual and equal trypsin- and chymotrypsin-like specificities. Particularly deleterious were β-branched side chains of Ile and Val. The P
1 substrate preferences found for cathepsin G are distinctly different from many other serine proteinases, including fiddler crab collagenase and chymotrypsin. The
k
cat/
K
m values obtained for P
1 Lys, Phe, Arg and Leu substrates correlate well with those determined for analogous P
1 mutants of basic pancreatic trypsin inhibitor (BPTI) obtained through recombinant techniques. To characterise the subsite specificity of the enzyme, a series of
Cucurbita maxima trypsin inhibitor I (CMTI I) mutants were used comprising P
2–P
3′ and P
12′ positions. All the mutants obtained were inhibitors of cathepsin G with association constants in the range: 10
5–10
9 M
−1. Some of the mutations destabilised complex formation. In particular, Met
8→Arg substitution at P
3′, which increased association constant for chymotrypsin 46-fold, led to a 7-fold decrease of binding with cathepsin G. In addition, mutation of Ile
6 at position P
1′ either to Val or Asp was deleterious for cathepsin G. In two cases (Ala
18→Gly (P
12′) and Pro
4→Thr (P
2)), about a 10-fold increase in association constants was observed.