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J. Biol. Chem., Vol. 276, Issue 18, 15025-15033, May 4, 2001

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Coiled Coil Region of Streptokinase -Domain Is Essential for Plasminogen Activation^*

Dung-Ho Wu, Guey-Yueh Shi, Woei-Jer Chuang, Jung-Mao Hsu, Kung-Chia Young^§, Chung-Wen Chang, and Hua-Lin Wu^¶

From the Departments of Biochemistry and ^§ Medical Technology, College of Medicine, National Cheng Kung University, Tainan, Taiwan 701 and  Department of Pharmacy, Chia Nan University of Pharmacy and Science, Tainan, Taiwan 710, Republic of China

The specific functions of the amino acid residues in the streptokinase (SK) -domain were analyzed by studying the interactions of human plasminogen (HPlg) and SK mutants prepared by charge-to-alanine mutagenesis. SK with mutations of groups of amino acids outside the coiled coil region of SK -domain, SK_{K278A,K279A,E281A,K282A}, and SK_D360A,R363A had similar HPlg activator activities as wild-type SK. However, significant changes of the functions of SK with mutations within the coiled coil region were observed. Both SK_{D322A,R324A,D325A} and SK_{R330A,D331A,K332A,K334A} had decreased amounts of complex formation with microplasminogen and failed to activate HPlg. SK_D328A,R330A had a 21-fold reduced catalytic efficiency for HPlg activation. The studies of SK with single amino acid mutation to Ala demonstrate that Arg³²⁴, Asp³²⁵, Lys³³², and Lys³³⁴ play important roles in the formation of a HPlg·SK complex. On the other hand, amino acid residues Asp³²², Asp³²⁸, and Arg³³⁰ of SK are involved in the virgin enzyme induction. Potential contact between Lys³³² of SK and Glu⁶²³ of human microplasmin and strong interactions between Asp³²⁸ and Lys³³⁰, Asp³³¹ and Lys³³⁴, and Asp³²² and Lys³³⁴ of SK are noticed. These interactions are important in maintaining a coiled coil conformation. Therefore, we conclude that the coiled coil region of SK -domain, SK(Leu³¹⁴-Ala³⁴²), plays very important roles in HPlg activation by participating in virgin enzyme induction and stabilizing the activator complex.

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