Human plasma kallikrein and tissue kallikrein binding to a substrate basedon the reactive site of a factor Xa inhibitor isolated from Bauhinia ungulata seeds

Kunitz type Bauhinia ungulata factor Xa inhibitor (BuXI) was purified from B. ungulata seeds. BuXI inactivates factor Xa and human plasma kallikrein ( HuPK) with K-i values of 18.4 and 6.9 nM, respectively. However, Bauhinia v ariegata trypsin inhibitor (BvTI) which is 70% homologous to BuXI does not inhibit factor Xa and is less efficient on HuPK (K-i = 80 nM). The comparis on between BuXI and BvTI reactive site structure indicates differences at M et(59), Thr(66) and Met(67) residues. The hydrolysis rate of quenched fluor escence peptide substrates based on BuXI reactive site sequence, Abz-VMIAAL PRTMFIQ-EDDnp (leading peptide), by HuPK and porcine pancreatic kallikrein (PoPK) is low, but hydrolysis is enhanced with Abz-VMIAALPRTMQ-EDDnp, deriv ed from the leading peptide shortened by removing the dipeptide Phe-Ileu fr om the C-terminal portion, for HuPK (K-m = 0.68 mu M, k(cat)/K-m = 1.3 X 10 (6) M-1 s(-1)), and the shorter substrate Abz-LPRTMQ-EDDnp is better for Po PK (K-m = 0.66 mu M, k(cat)/K-m = 2.2 X 10(3) M-1 s(-1)). The contribution of substrate methionine residues to HuPK and PoPK hydrolysis differs from t hat observed with factor Xa. The determined K-m and k(cat) values suggest t hat the substrates interact with kallikreins the same as an enzyme and inhi bitor interacts to form complexes. (C) 1999 Elsevier Science B.V. All right s reserved.