Structure-function analysis of the streptokinase amino terminus (residues 1-59).
Streptokinase (SK) binds to plasminogen (Pg) to form a complex that converts substrate Pg to plasmin. Residues 1-59 of SK regulate its capacity to induce an active site in bound Pg by a nonproteolytic mechanism and to activate substrate Pg in a fibrin-independent manner. We analyzed 24 SK mutants to better define the functional properties of SK-(1-59). Mutations within the alphabeta1 strand (residues 17-26) of SK completely prevented nonproteolytic active site induction in bound Pg and rendered SK incapable of protecting plasmin from inhibition by alpha2-antiplasmin. However, when fibrin-bound, the activities of alphabeta1 strand mutants were similar to that of wild-type (WT) SK and resistant to alpha2-antiplasmin. Mutation of Ile1 of SK also prevented nonproteolytic active site induction in bound Pg. However, unlike alphabeta1 strand mutants, the functional defect of Ile1 mutants was not relieved by fibrin, and complexes of Ile1 mutants and plasmin were resistant to alpha2-antiplasmin. Plasmin enhanced the activities of alphabeta1 strand and Ile1 mutants, suggesting that SK-plasmin complexes activated mutant SK.Pg complexes by hydrolyzing the Pg Arg561-Val562 bond. Mutational analysis of Glu39 of SK suggested that a salt bridge between Glu39 and Arg719 of Pg is important, but not essential, for nonproteolytic active site induction in Pg. Deleting residues 1-59 rendered SK dependent on plasmin and fibrin to generate plasminogen activator (PA) activity. However, the PA activity of SK-(60-414) in the presence of fibrin was markedly reduced compared with WT SK. Despite its reduced PA activity, the fibrinolytic potency of SK-(60-414) was greater than that of WT SK at higher (but not lower) SK concentrations due to its capacity to deplete plasma Pg. These studies define mechanisms by which the SK alpha domain regulates rapid active site induction in bound Pg, contributes to the resistance of the SK-plasmin complex to alpha2-antiplasmin, and controls fibrin-independent Pg activation.
Mundada
Lakshmi V
LV
Research Service, Ann Arbor Veterans Affairs Hospital and the Department of Internal Medicine, University of Michigan Medical School, Ann Arbor 48109, USA.
Prorok
Mary
M
DeFord
Melanie E
ME
Figuera
Mariana
M
Castellino
Francis J
FJ
Fay
William P
WP
D020134:Catalytic Domain / Q000235:genetics; D009154:Mutation; D013291:Streptococcus / Q000201:enzymology; D013300:Streptokinase / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics
D013300:Streptokinase