Role of the N-terminal region of staphylokinase (SAK): evidence for the participation of the N-terminal region of SAK in the enzyme-substrate complexformation

Staphylokinase (SAK) forms an inactive 1:1 complex with plasminogen (PG), w hich requires both the conversion of PG to plasmin (Pm) to expose an active site in PG-SAK activator complex and the amino-terminal processing of SAK to expose the positively charged (Lys-11) amino-terminus after removal of t he 10 N-terminal amino acid residues from the full length protein. The mech anism by which the N-terminal segment of SAK affects its PG activation capa bility was investigated by generating SAK mutants, blocked in the native am ino-terminal processing site of SAK, and carrying an alteration in the plac ement of the positively charged amino acid residue, Lys-11, and further stu dying their interaction with PG, Pm, miniplasmin and kringle structures. A ternary complex formation between PG-SAK PG was observed when an immobilize d PG-SAR binary complex interacted with free radiolabelled PG in a sandwich binding experiment. Formation of this ternary complex was inhibited by a l ysine analog, 6-aminocaproic acid (EACA), in a concentration dependent mann er, suggesting the involvement of lysine binding site(s) in this process. I n contrast, EACA did not significantly affect the formation of binary compl ex formed by native SAK or its mutant derivatives. Furthermore, the binary (activator) complex formed between PG and SAK mutant, PRM3, lacking the N-t erminal lysine 1, exhibited 3-4-fold reduced binding with PG, Pm or minipla smin substrate during ternary complex formation as compared to native SAI(. Additionally, activator complex formed with PRM3 failed to activate minipl asminogen and exhibited highly diminished activation of substrate PG. Prote in binding studies indicated that it has 3-5-fold reduction in ternary comp lex formation with miniplasmin but not with the kringle structure. In aggre gate, these observations provide experimental evidence for the participatio n of the N-terminal region of SAK in accession and processing of substrate by the SAK-Pm activator complex to potentiate the PG activation by enhancin g and/or stabilizing the interaction of free PG. (C) 2000 Federation of Eur opean Biochemical Societies.