title: Functional roles of streptokinase C-terminal flexible peptide in active site formation and substrate recognition in plasminogen activation.
abstract: The bacterial protein streptokinase (SK) activates human plasminogen (Pg) into the fibrinolytic protease plasmin (Pm). Roughly 40 residues from the SK C-terminal domain are mobile in the crystal structure of SK complexed with the catalytic domain of Pm, and the functions of this C-tail remain elusive. To better define its roles in Pg activation, we constructed and characterized three C-terminal truncation mutants containing SK residues 1-378, 1-386, and 1-401, respectively. They exhibit gradually reduced amidolytic activity and Pg-activator activity, as well as marginally decreased binding affinity toward Pg, as more of the C-terminus is deleted. As compared with full-length SK, the shortest construct, SK(1-378), exhibits an 80% decrease in amidolytic activity (k(cat)/K(M)), an 80% decrease in Pg-activator activity, and a 30% increase in the dissociation constant toward the Pg catalytic domain. The C-terminal truncation mutations did not attenuate the resistance of the SK-Pm complex to alpha(2)-antiplasmin. Attempts at using a purified C-tail peptide to rescue the activity loss of the truncation mutants failed, suggesting that the integrity of the SK C-terminal peptide is important for the full function of SK.
authors.affiliation: Crystallography Research Program, Oklahoma Medical Research Foundation, 825 Northeast 13th Street, Oklahoma City, Oklahoma 73104, USA.
authors.forename: Peng
authors.initials: P
authors.lastname: Zhai
authors.forename: Nancy
authors.initials: N
authors.lastname: Wakeham
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Loy
authors.forename: Xuejun C
authors.initials: XC
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D004789:Enzyme Activation; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D010958:Plasminogen / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D010960:Plasminogen Activators / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry; D017384:Sequence Deletion; D013291:Streptococcus / Q000201:enzymology / Q000235:genetics; D013300:Streptokinase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D013379:Substrate Specificity / Q000235:genetics; D000979:alpha-2-Antiplasmin / Q000737:chemistry
chemical_list: D009097:Multienzyme Complexes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000979:alpha-2-Antiplasmin; C111879:microplasminogen protein, human; D010958:Plasminogen; D013300:Streptokinase; D010960:Plasminogen Activators