Role of the 88-97 loop in plasminogen activation by streptokinase probed through site-specific mutagenesis.
The role of a prominent surface-exposed loop (residues 88-97) in the alpha domain of streptokinase (SK), in human plasminogen (HPG) activation was explored through its selective mutagenesis and deletion studies. We first made a conformationally constrained derivative of the loop by the substitution of sequences known to possess a strong propensity for beta-turn formation. The mutant so formed (termed SK88-97-Beta Turn), when tested for co-factor activity against substrate HPG, after first forming a 1:1 molar complex with human plasmin (HPN), showed a nearly 6-fold decreased co-factor activity compared to the wild-type, native SK. The major catalytic change was observed to be at the k(cat) level, with relatively minor changes in Km values against HPG. Real-time binary interaction (i.e. the 1:1 complexation between SK, or its mutant/s, with HPG), and ternary complexation studies (i.e. the docking of a substrate HPG molecule into the preformed SK-HPG complex) using Surface Plasmon Resonance were done. These studies revealed minor alterations in binary complex formation but the ternary interactions of the substitution and/or deletion mutants were found to be decreased for full-length HPG compared to that for native SK.HPG. In contrast, their ternary interactions with the isolated five-kringle domain unit of plasminogen (K1-5) showed Kd values comparable to that seen with the native SK.HPG complex. Taking into consideration the overall alterations observed in catalytic levels after site-specific mutagenesis and complete loop deletion of the 88-97 loop, on the one hand, and its known position at the SK-HPG interface in the binary complex, suggests the importance of this loop. The present results suggest that the 88-97 loop of the alpha domain of SK contributes towards catalytic turn-over, even though its individual contribution towards enzyme-substrate affinity per se is minimal.
Yadav
Suman
S
Institute of Microbial Technology (C.S.I.R), Sector 39-A, Chandigarh-160036, India.
Datt
Manish
M
Singh
Balvinder
B
Sahni
Girish
G
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010958:Plasminogen / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013300:Streptokinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
D017931:DNA Primers; D011994:Recombinant Proteins; D010958:Plasminogen; D013300:Streptokinase