Roles of the P1, P2, and P3 residues in determining inhibitory specificityof kallistatin toward human tissue kallikrein

Kallistatin is a serpin with a unique P1 Phe, which confers an excellent in hibitory specificity toward tissue kallikrein, In this study, we investigat ed the P3-P2-P1 residues (residues 386-388) of human kallistatin in determi ning inhibitory specificity toward human tissue kallikrein by site-directed mutagenesis and molecular modeling. Human kallistatin mutants with 19 diff erent amino acid substitutions at each P1, P2, or P3 residue were created a nd purified to compare their kallikrein binding activity. Complex formation assay showed that P1 Arg, P1 Phe (wild type), P1 Lys, PI Tyr, PI Met, and P1 Leu display significant binding activity with tissue kallikrein among th e P1 variants. Kinetic analysis showed the inhibitory activities of the P1 mutants toward tissue kallikrein in the order of P1 Arg > P1 Phe > P1 Lys g reater than or equal to PI Tyr > P1 Leu greater than or equal to P1 Met. P1 Phe displays a better selectivity for human tissue kallikrein than P1 Arg, since P1 Arg also inhibits several other serine proteinases, Heparin disti nguishes the inhibitory specificity of kallistatin toward kallikrein versus chymotrypsin, For the P2 and P3 variants, the mutants with hydrophobic and bulky amino acids at P2 and basic amino acids at P3 display better binding activity with tissue kallikrein, The inhibitory activities of these mutant s toward tissue kallikrein are in the order of P2 Phe (wild type) > P2 Leu > P2 Trp > P2 Met and P3 Arg > P3 Lys (wild type). Molecular modeling of th e reactive center loop of kallistatin bound to the reactive crevice of tiss ue kallikrein indicated that the P2 residue required a long and bulky hydro phobic side chain to reach and fill the hydrophobic S2 cleft generated by T yr(99) and Trp(219) Of tissue kallikrein. Basic amino acids at P3 could sta bilize complex formation by forming electrostatic interaction with Asp(98J) and hydrogen bond with Gln(174) of tissue kallikrein, Our results indicate that tissue kallikrein is a specific target proteinase for kallistatin.