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 Arg389 and Ser390) is a typical cleavage point for a trypsin-like enzyme whereas site II (between Met379 and Lys380) is unusual and unique to TK. In order to learn more about the structural requirements and mechanism of cleavage at site II, we studied the hydrolysis by TK of several synthetic LK fragments varying in length between 4 and 22 residues and containing either site II only or both sites I and II. 2. Blocking site I cleavage in LK fragments by substituting DArg for LArg at position 389 or omitting site I from the sequence still allowed cleavage to proceed at site II. Replacement or deletion of selected amino acid residues in these fragments demonstrated that the presence of Arg381 was essential for site II cleavage to occur whereas Pro383, Phe385 and Ser386 could be replaced with Ala without affecting binding or cleavage by TK. Ki values towards TK were determined for all LK fragments in order to compare their binding affinities to the enzyme. Short peptides containing site II only exhibited high Ki values (> or = 100 microM) whereas longer fragments containing both sites I and II had Ki values of 2-7 microM. 3. In order to bring sites I and II into close proximity spatially and thus facilitating efficient cleavage in the enzyme-substrate complex, we prepared several cyclic analogs of the longer LK fragments.(ABSTRACT TRUNCATED AT 250 WORDS)
Sueiras-Diaz
J
J
Ferring Research Institute, University of Southampton Research Centre, Chilworth, UK.
Jones
D M
DM
Ashworth
D
D
Horton
J
J
Evans
D M
DM
Szelke
M
M
D000595:Amino Acid Sequence; D001665:Binding Sites; D006801:Humans; D006868:Hydrolysis; D007609:Kallidin / Q000378:metabolism; D007610:Kallikreins / Q000378:metabolism*; D007704:Kininogens / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D011487:Protein Conformation; D020840:Tissue Kallikreins
D007704:Kininogens; D010446:Peptide Fragments; D007609:Kallidin; D007610:Kallikreins; D020840:Tissue Kallikreins