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Biochim Biophys Acta. 1998 Jul 28;1386(1):189-98.

Specificity of human cathepsin G.

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Institute of Biochemistry and Molecular Biology, University of Wroclaw, Tamka 2, 50-137 Wroclaw, Poland.


A series of tetrapeptide p-nitroanilide substrates of the general formula: suc-Ala-Ala-Pro-Aaa-p-nitroanilide was used to map the S1 binding pocket of human cathepsin G. Based on the kcat/Km 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 beta-branched side chains of Ile and Val. The P1 substrate preferences found for cathepsin G are distinctly different from many other serine proteinases, including fiddler crab collagenase and chymotrypsin. The kcat/Km values obtained for P1 Lys, Phe, Arg and Leu substrates correlate well with those determined for analogous P1 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 P2-P3' and P12' positions. All the mutants obtained were inhibitors of cathepsin G with association constants in the range: 105-109 M-1. Some of the mutations destabilised complex formation. In particular, Met8-->Arg substitution at P3', which increased association constant for chymotrypsin 46-fold, led to a 7-fold decrease of binding with cathepsin G. In addition, mutation of Ile6 at position P1' either to Val or Asp was deleterious for cathepsin G. In two cases (Ala18-->Gly (P12') and Pro4-->Thr (P2)), about a 10-fold increase in association constants was observed.

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