DETERMINANTS OF THE UNUSUAL CLEAVAGE SPECIFICITY OF LYSYL-BRADYKININ-RELEASING KALLIKREINS

Kinetic data for the hydrolysis by human tissue kallikrein of fluoroge nic peptides with o-aminobenzoyl-Phe-Arg (Abz-FR) as the acyl group an d different leaving groups demonstrate that interactions with the S'(1 ), S'(2), and S'(3), subsites are important for cleavage efficiency. I n addition, studies on the hydrolysis of fluorogenic peptides with the human kininogen sequence spanning the scissile Met-Lys bond -S-L-M-K- R-P-N-(2,4-dinitrophenyl)ethylenediamine] and analogues with different residues at positions P'(1), P'(2) and P'(3) showed that (a) the pres ence of a proline residue at P'(3) and the interactions with the tissu e kallikrein-binding sites S-2 to S'(2) are determinants of Met-Lys bo nd cleavage and (b) residues P-3, P-4 and/or P-5 are important for cle avage efficiency. The substitution of phenylalanine for methionine or arginine in substrates with scissile Met-Lys or Arg-Xaa bonds demonstr ated that lysyl-bradykinin-releasing tissue kallikreins also have a pr imary specificity for phenylalanine. The replacement of arginine by ph enylalanine in (D)P-F-R-p-nitroanilide (pNA) produced an efficient and specific chromogenic substrate, (D)P-F-F-pNA, for the lysyl-bradykini n-releasing tissue kallikreins as it is resistant to plasma kallikrein and other arginine hydrolases.