Specificity of human cathepsin G

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 P-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. (C) 1998 Elsevier Science B.V. All rights reserved.