鏈激脢 ( streptokinase, SK ) 是人類血纖維蛋白溶脢原（human plasminogen, HPlg）的活化子，含有414個胺基酸，分子量約45kDa的單一 鏈，由β-溶血性鏈球菌所分泌的菌體外蛋白質。SK能與HPlg形成莫爾比1:1之活化複合體，此複合體能水解游離態HPlg中Arg561-Val562間的 鏈，而將游離態的HPlg活化為HPlm；新生的Val562 N端會與Asp740形成salt bridge，造成活化區構型的改變，使HPlm具有蛋白水解脢的活性，此過程類似胰蛋白脢原(trypsinogen)活化成胰蛋白脢(trypsin)。活化的HPlm能水解血纖維蛋白，造成血塊的溶解。

SK誘導HPlg出現”virgin enzyme”活性的過程，並不會水解Arg561-Val562間的 鏈。SK活化HPlg的機制目前尚未清楚，有一”binding activation”假說認為，當SK與HPlg結合時，SK的γ定義區能與HPlg的autolysis loop交互作用，造成Lys698構型的改變，使Lys698與Asp740產生salt bridge，進而促成活化區的暴露。然而另有研究顯示，SK的Ile1才是與Asp740形成salt bridge的重要胺基酸，當SK缺少了Ile1，HPlg的活化區便無法形成，且SK之N端 鏈能穩定活化複合體；後者被稱為molecular sexuality 假說。

為釐清SK Ile1及N端 鏈在HPlg活化中所扮演的角色。本實驗室構築且純化出SK重組蛋白SK (1-378)、SK (2-378)、SK (16-378)、SK (Gly-1-378)、SK (Ile-Tag-16-378)(N端胺基酸定序分析90%的SK (1-378)和SK (Ile-Tag-16-378)在Ile前另有Met)。比活性及HPlg活化試驗中，所有SK突變株均能如原型SK有效活化HPlg。酵素動力學試驗顯示，各突變株的kcat和Km值沒有明顯差異。當以莫爾濃度比SK:HPlg=6:1、 4:1、 2:1或1:1之SK誘導HPlg的醯胺水解活性(amidolytic activity)時，HPlg的醯胺水解活性的誘導會隨著SK濃度的增加而受到延滯，但是原型SK (native SK)和SK (1-378)則無此現象。4-methylumbelliferyl p-guanidinobenzoate(MUGB) titration實驗顯示:等莫爾濃度下，各突變株均能使HPlg產生活化區。以非連續法分析HPlg之virgin enzyme活性，發現除了原型SK及SK (1-378)，當過量的SK越多時，virgin enzyme活性也會越晚出現。

以催化濃度的SK活化HPlg時，缺少了N端序列或以Gly遮蔽Ile1之N端amino group並不影響SK活化HPlg。然而以過量的SK誘導HPlg活化時，隨著N端胺基酸截短的越多，HPlg的活化會有越長的延滯期，而在SK (16-378)的N端接上T7∙Tag，能減少延滯期的時間，顯示SK的N端在過量SK誘導HPlg活化時才扮演重要的角色，但此一交互作用的專一性並不高，因為與SK(1-15)無同源性的序列T7∙Tag亦能部分取代SK (1-15)的功能。此外SK (1-378)在N端多了Met，但是其活化HPlg的表現仍與原型SK相似，而SK (Gly-1-378)卻有延滯期的出現，顯示第一胺基酸不一定得是Ile，Met也能取代Ile的角色，但是中性的Gly便無法取代其功能。由以上結果推論，SK的N端序列在誘導HPlg構形活化上並不扮演重要的角色，但以過量SK誘導HPlg構型活化時，SK的N端可能與μ-HPlg作用，保護活化區不受過量SK的干擾，進而加速HPlg的構型活化。

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 uggested 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.

Bajaj S. P., Castellino F. J. Activation of plasminogen by equimolar level of streptokinase. J. Biol. Chem. 252, 492-498(1977).

Ben-Bassat A., Bauer K., Chang S. Y., Myambo K., Boosman A., Chang S. Precessing of the initiation Met from proteins: properties of the Escherichia coli Met aminopeptidase and its gene structure. J. Bacteriol 169, 751-757(1978).

Bode W., Huber R. Induction of the bovine trypsinogen-trypsin transition by peptides sequentially similar to the N-terminus of trypsin. FEBS Lett. 68, 231-271(1976).

Boxrud P. D., Verhamme I. M. A., Fay W. P., Bock P. E. Streptokinase triggers conformational activation of plasminogen through specific interactions of the aminol-terminal sequence and stabilizes the active zymogen conformation. J. Biol. Chem. 276, 26084-26089(2001).

Castellino F. J. An unique enzyme protein substrate modified reaction: plasmin streptokinase interaction. Trends Biochem. Sci. 4, 1-5(1979).

Esmon C. T., Mather T. Switching serine protease specificity. Nat. Struct. Biol 5, 933-937(1998).

Jandl J. M. Textbook of hematology. 1st ed, boston: Little Brown (1987).

Loy J. A., Lin X., Schenone M., Castellino F. J., Zhang X. C., Tang J. Domain interaction between streptokinase and human plasminogen. Biochemistry 40, 14686-14695(2001).

Mangel W. F., Lin B., Ramafrishnan V. Characterization of an extremely large, ligand-induced conformational change in plasminogen. Science 248, 69-73(1990).

McClintock D. H., Bell D. H. The mechanism of activation of human plasminogen by streptokinase. Biochem Biophys Res Commun. 43, 694-702(1971).

Miyashita C., Wenzel E., Heiden M. Plasminogen: a brief introduction into biochemistry and function. Haemostasis. 18, suppl. 1, 7-13(1988).

Petersen T. E., Martzen M. R., Ichinose A., Davie E. W. Characterization of the gene for plasminogen, a key proenzyme in the fibrinolytic system. J. Biol. Chem. 265, 6104-6111(1990).

Reddy K. N. N. Streptokinase-biochemistry and clinical application. Enzyme 40, 79-89(1988).

Reed G. L., Lin L. F., Parhami-Seren B., Kussie P. Identification of a plasminogen binding region in streptokinase that is necessary for the creation of a functional streptokinase-plasminogen activator complex. Biochemistry. 34, 10266-10271(1995).

Shi G. Y., Chang B. I., Chen S. M., Wu D. H., Wu H. L. Function of streptokianse fragments in plasminogen activation. J. Biol. Chem. 304, 235-241(1994).

Shi G. Y., Chang B. I., Wu D. H., Ha Y. M. Activation of human and bovine plasminogen by the microplasmin and streptokinase complex. Thromb. Res 58, 317-329(1990).

Shi G. Y., Wu H. L. Isolation and characterization of microplasminogen. J. Biol. Chem. 263, 17071-17075(1988).

Tomar R. H., Taylor F. B. The streptokinase-human plasminogen activator complex. Biochem. J. 125, 739-802(1971).

Tsunasawa S., Stewart J. W., Sherman F. Amino-terminal precessing of the mutant forms of yeast iso-1-cytochrome c: the specificities of metionine aminopeptidase and acetyltrasnferase. J. Biol. Chem. 260, 5382-5391(1985).

Wang S., Reed G. L., Hedstrom L. Deletion of Ile1 changes the mechanism of streptokinase: evidence for the molecular sexuality hypothesis. Biochemistry 38, 5232-5240(1999).

Wang S., Reed G. L., Hedstrom L. Zymogen activation in the streptokinase-plasminogen complex. Ile1 is required for the formation of a functional active site. Eur. J. Biochem. 267, 3994-4001(2000)

Wang X., Lin X., Loy J. A., Tang J., Zhang X. Crystal structure of the catalytic domain of human plasmin complexed with streptokinase. Science 281,1662-1665(1998).

Whol R. C., Summria L., Robbin K. C. Kinetics of activation of human plasminogen by different activator species at pH 7.4 and 37℃. J. Biol. Chem. 255, 2005-2013(1980).

Wu H. L., Shi G. Y., Wohl R. C., Bender M. L. Preparation and purification of microplasmin. Proc. Natl. Acad. Sci. U.S.A. 84, 8292-8295(1987).

Wu H. L., Shi G. Y., Wohl R. C., Bender M. L. Structure and formation of microplasmin. Proc. Natl. Acad. Sci. U.S.A. 84, 8793-8795(1987).

Wu D. H., Shi G. Y., Chuang W. J., Hsu J. M., Young K. C., Chang C. W., Wu H. L. Coiled coil region of streptokinase gamma-domain is essential for plasminogen activation. J. Biol. Chem. 276, 15025-15033(2001).

Young K. C., Shi G. Y., Chang Y. F., Chang B. I., Chang L. C., Lai M. D., Chuang W. J., Wu H. L. Interaction of streptokinase and plasminogen. J. Biol. Chem. 270, 29601-29606(1995).

Young K. C., Shi G. Y., Wu D. H., Chang L. C., Chang B. I., Ou C. P., Wu H. L. Plasminogen activation by streptokinase via a unigue mechanism. J. Biol. Chem. 273, 3110-3116(1998)