Summary: | 碩士 === 國立成功大學 === 生物化學研究所 === 90 === Streptokinase (SK) is a single-peptide secretory protein of 414 amino acid residues produced by various strains of β-hemolytic Streptococcus. The SK and human plasminogen (HPlg) can form an equimolar activator complex that catalyzes the cleavage of the Agr561-Val562 peptide bond of HPlg to human plasmin (HPlm); the newly formed N-terminus of Val562 is believed to insert inwardly to form a salt bridge with the carboxylate of Asp740 in a manner analogous to the activation of trypsinogen to trypsin. Plm is a potent protease that in turn catalyzes the hydrolysis of fibrin, which causes the dissolution of blood clot.
By an unclearly-identified mechanism, SK can activate HPlg protease activity without the proteolytic cleavage. The “binding activation” hypothesis suggests that the binding of SK γdomain to the autolysis loop region of HPlg may cause a conformation change of Lys698, resulting in the formation of the critical salt bridge with Asp740 and thereby the activation of HPlg catalytic apparatus. However, recent reports have proposed that the N-terminus of SK might directly insert into μPlg domain to form salt linkage with Asp740, which was called “molecular sexuality” hypothesis.
In order to examine the function of Ile 1 and the N-terminal peptide of SK in the conformational activation of HPlg, we designed various SK mutants, including SK (1-378), SK (2-378), SK (16-378), SK (Gly-1-378) (with Gly residure to block the N-terminus of Ile1), and SK (Ile-Tag-16-378) (with Ile residue in the N-terminus); N-terminal amino acid sequencing revealed that SK (1-378) and SK (Ile-Tag-16-378) contain 90% N-terminal Met. According to specific activity and HPlg activation assays, all SK mutants could activate HPlg as efficiently as native SK with a catalytic concentration of SK. The kcat and Km values among those mutants were also similar. However, when the amount of SK is more than HPlg, the appearance of amidolytic activity was delayed in all the SK mutants except native SK and SK (1-378). The active site titration of HPlg with 4-methylumbelliferyl p-guanidinobenzoate(MUGB) revealed that all mutants could generate an active site in HPlg in an equimolar mixture, and that without free N-terminus or the Ile1 residue of SK , the exposure of active site was significantly inhibited under excess SK condition. Discontinuous amidolytic activity assays indicated that deletion or capping in the SK N-terminus would cause the delay of exposure of active site in HPlg.
Ile1 of SK may not play an critical role in the activation of HPlg with catalytic amount of SK, because the N-terminal deletion and the N-terminal blocking with Gly did not affect the activity of SK mutants. However, in the activation of HPlg with excess SK, the N-terminal deletion and the N-termial blocking with Gly would cause the delay of the HPlg conformational activation, and adding T7∙Tag in the N-terminal of SK (16-378) could decrease this effect. It was auggested that SK N-terminal sequence may be important in the HPlg activation with excess SK, and that the interaction of SK (1-15) with HPlg would not be highly specific, because even the T7∙Tag could subtitude the function of SK (1-15). Bescides, althought SK (1-378) had initiation Met in the N-terminal, the behavior was similar as native SK, but SK (Gly-1-378) had delay time in induction of HPlg conformaitonal activation. This result implied that Ile1 may not be an critital residue for initiation activation of HPlg, but a hydrophobic residue such as Met could be in place of the function of Ile1.
To sum up those observations, Ile1 of SK would not be an significant residue in the activation of HPlg with catalytic amount of SK. But when SK is over than HPlg, the excess SK may influence the formation of the active SK-HPlg complex, and the N-terminal sequence of SK could prevent the interaction of excess to SK-HPlg complex, therefore promoting the formation of SK-HPlg virgin enzyme activity.
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