Affiliation:
1. Institute of Microbial Technology (IMTECH), Sector 39 A, Chandigarh-160036, India
Abstract
Staphylokinsae (SAK) forms a bimolecular complex with human plasmin(ogen) and changes its substrate specificity by exposing new exosites that enhances accession of substrate plasminogen (PG) to the plasmin (Pm) active site. Protein modelling studies indicated the crucial role of a loop in SAK (SAK 90-loop; Thr90—Glu100) for the docking of the substrate PG to the SAK—Pm complex. Function of SAK 90-loop was studied by site-directed mutagenesis and loop deletion. Deletion of nine amino acid residues (Tyr92—Glu100) from the SAK 90-loop, resulted in ≈60% reduction in the PG activation, but it retained the ability to generate an active site within the complex of loop mutant of SAK (SAKΔ90) and Pm. The preformed activator complex of SAKΔ90 with Pm, however, displayed a 50–60% reduction in substrate PG activation that remained unaffected in the presence of kringle domains (K1+K2+K3+K4) of PG, whereas PG activation by SAK—Pm complex displayed ∼50% reduction in the presence of kringles, suggesting the involvement of the kringle domains in modulating the PG activation by native SAK but not by SAKΔ90. Lysine residues (Lys94, Lys96, Lys97 and Lys98) of the SAK 90-loop were individually mutated into alanine and, among these four SAK loop mutants, SAKK97A and SAKK98A exhibited specific activities about one-third and one-quarter respectively of the native SAK. The kinetic parameters of PG activation of their 1:1 complex with Pm indicated that the Km values of PG towards the activator complex of these two SAK mutants were 4–6-fold higher, suggesting the decreased accessibility of the substrate PG to the activator complex formed by these SAK mutants. These results demonstrated the involvement of the Lys97 and Lys98 residues of the SAK 90-loop in assisting the interaction with substrate PG. These interactions of SAK—Pm activator complex via the SAK 90-loop may provide additional anchorage site(s) to the substrate PG that, in turn, may promote the overall process of SAK-mediated PG activation.
Subject
Cell Biology,Molecular Biology,Biochemistry
Cited by
22 articles.
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