Specificity of human cathepsin G

Abstract

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.