title: Role of the N-terminal region of staphylokinase (SAK): evidence for the participation of the N-terminal region of SAK in the enzyme-substrate complex formation.
abstract: Staphylokinase (SAK) forms an inactive 1:1 complex with plasminogen (PG), which 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 the 10 N-terminal amino acid residues from the full length protein. The mechanism by which the N-terminal segment of SAK affects its PG activation capability was investigated by generating SAK mutants, blocked in the native amino-terminal processing site of SAK, and carrying an alteration in the placement of the positively charged amino acid residue, Lys-11, and further studying their interaction with PG, Pm, miniplasmin and kringle structures. A ternary complex formation between PG-SAK PG was observed when an immobilized PG-SAK binary complex interacted with free radiolabelled PG in a sandwich binding experiment. Formation of this ternary complex was inhibited by a lysine analog, 6-aminocaproic acid (EACA), in a concentration dependent manner, suggesting the involvement of lysine binding site(s) in this process. In contrast, EACA did not significantly affect the formation of binary complex formed by native SAK or its mutant derivatives. Furthermore, the binary (activator) complex formed between PG and SAK mutant, PRM3, lacking the N-terminal lysine 11, exhibited 3-4-fold reduced binding with PG, Pm or miniplasmin substrate during ternary complex formation as compared to native SAK. Additionally, activator complex formed with PRM3 failed to activate miniplasminogen and exhibited highly diminished activation of substrate PG. Protein binding studies indicated that it has 3-5-fold reduction in ternary complex formation with miniplasmin but not with the kringle structure. In aggregate, these observations provide experimental evidence for the participation 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 enhancing and/or stabilizing the interaction of free PG.
authors.affiliation: Division of Molecular Biology, Institute of Microbial Technology, Sector 39A, 160036, Chandigarh, India.
authors.forename: G
authors.initials: G
authors.lastname: Rajamohan
authors.forename: K L
authors.initials: KL
authors.lastname: Dikshit
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D015119:Aminocaproic Acid / Q000494:pharmacology; D001665:Binding Sites; D002384:Catalysis / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005341:Fibrinolysin / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D018082:Kringles; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010958:Plasminogen / Q000737:chemistry / Q000378:metabolism; D010960:Plasminogen Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011499:Protein Processing, Post-Translational; D013211:Staphylococcus aureus / Q000201:enzymology*
chemical_list: D007457:Iodine Radioisotopes; D010446:Peptide Fragments; C019493:miniplasmin; D010958:Plasminogen; D010960:Plasminogen Activators; D005341:Fibrinolysin; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus; D008239:Lysine; D015119:Aminocaproic Acid