Human plasma kallikrein and tissue kallikrein binding to a substrate based on 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 variegata 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 comparison between BuXI and BvTI reactive site structure indicates differences at Met(59), Thr(66) and Met(67) residues. The hydrolysis rate of quenched fluorescence peptide substrates based on BuXI reactive site sequence, Abz-VMIAALPRTMFIQ-EDDnp (leading peptide), by HuPK and porcine pancreatic kallikrein (PoPK) is low, but hydrolysis is enhanced with Abz-VMIAALPRTMQ-EDDnp, derived from the leading peptide shortened by removing the dipeptide Phe-Ileu from 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 PoPK (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 that observed with factor Xa. The determined K-m and k(cat) values suggest that the substrates interact with kallikreins the same as an enzyme and inhibitor interacts to form complexes. (C) 1999 Elsevier Science B.V. All rights reserved.