CLEAVAGE OF HUMAN KININOGEN FRAGMENTS AT MET-LYS BY HUMAN TISSUE KALLIKREIN

1. Tissue kallikrein (TK) cleaves low molecular weight kininogen (LK) at two sites to release kallidin: site I (between Arg(389) and Ser(390 )) is a typical cleavage point for a trypsin-like enzyme whereas site II (between Met(379) and Lys(380)) is unusual and unique to TK. In ord er to learn more about the structural requirements and mechanism of cl eavage al site II, we studied the hydrolysis by TK of several syntheti c LK fragments varying in length between 4 and 22 residues and contain ing either site II only or both sites I and II. 2. Blocking site I cle avage in LK fragments by substituting DArg for LArg at position 389 or omitting site I from the sequence still allowed cleavage to proceed a t site II. Replacement or deletion of selected amino acid residues in these fragments demonstrated that the presence of Arg(381) was essenti al for site II cleavage to occur whereas Pro(383), Phe(385) and Ser(38 6) could be replaced with Ala without affecting binding or cleavage by TK. Ki values towards TK were determined for all LK fragments in orde r to compare their binding affinities to the enzyme. Short peptides co ntaining site II only exhibited high Ki values ( greater than or equal to 100 mu M) whereas longer fragments containing both sites I and II had Ki values of 2-7 mu M. 3. In order to bring sites I and II into cl ose proximity spatially and thus facilitating efficient cleavage in th e enzyme-substrate complex, we prepared several cyclic analogs of the longer LK fragments. One of these cyclic peptides exhibited Ki = 0.9 m u M towards TK, indicating that the conformational change brought abou t by cyclisation facilitated binding to the enzyme. However, nuclear m agnetic resonance studies of a 54-residue LK fragment failed to demons trate a single dominant conformation in which sites I and II were clos e to each other.