S1′ and S2′ subsite specificities of human plasma kallikrein and tissue kallikrein 1 for the hydrolysis of peptides derived from the bradykinin domain of human kininogen

The S1′ and S2′ subsite specificities of human tissue kallikrein 1 (KLK1) and human plasma kallikrein (HPK) were examined with the peptide series Abz-GFSPFRXSRIQ-EDDnp and Abz-GFSPFRSXRIQ-EDDnp [X=natural amino acids or S(PO3H2)]. KLK1 efficiently hydrolyzed most of the peptides except those containing negatively charged amino acids at P1′ and P2′ positions. Abz-GFSPFRSSRIQ-EDDnp, as in human kininogen, is the best substrate for KLK1 and exclusively cleaved the R-S bond. All other peptides were cleaved also at the F-R bond. The synthetic human kininogen segment Abz-MISLMKRPPGFSPFRS390S391RI-NH2 was hydrolyzed by KLK1 first at R-S and then at M-K bonds, releasing Lys-bradykinin. In the S390 and S391 phosphorylated analogs, this order of hydrolysis was inverted due to the higher resistance of the R-S bond. Abz-MISLMKRPPG-FSPFRSS(PO3H2)391RI-NH2 was hydrolyzed by KLK1 at M-K and mainly at the F-R bond, releasing des-(Arg9)-Lys-Bk which is a B1 receptor agonist. HPK cleaved all the peptides at R and showed restricted specificity for S in the S1′ subsite, with lower specificity for the S2′ subsite. Abz-MISLMKRPPGFSPFRSSRI-NH2 was efficiently hydrolyzed by HPK under bradykinin release, while the analogs containing S(PO3H2) were poorly hydrolyzed. In conclusion, S1′ and S2′ subsite specificities of KLK1 and HPK showed peculiarities that were observed with substrates containing the amino acid sequence of human kininogen.