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J. Biol. Chem., Vol. 279, Issue 11, 10103-10108, March 12, 2004

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Residue Asp-189 Controls both Substrate Binding and the Monovalent Cation Specificity of Thrombin^*

Swati Prasad, Angelene M. Cantwell, Leslie A. Bush, Peter Shih, Hong Xu, and Enrico Di Cera

From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110

Residue Asp-189 plays an important dual role in thrombin: it defines the primary specificity for Arg side chains and participates indirectly in the coordination of Na⁺. The former role is shared by other proteases with trypsin-like specificity, whereas the latter is unique to Na⁺-activated proteases in blood coagulation and the complement system. Replacement of Asp-189 with Ala, Asn, Glu, and Ser drastically reduces the specificity toward substrates carrying Arg or Lys at P1, whereas it has little or no effect toward the hydrolysis of substrates carrying Phe at P1. These findings confirm the important role of Asp-189 in substrate recognition by trypsin-like proteases. The substitutions also affect significantly and unexpectedly the monovalent cation specificity of the enzyme. The Ala and Asn mutations abrogate monovalent cation binding, whereas the Ser and Glu mutations change the monovalent cation preference from Na⁺ to the smaller cation Li⁺ or to the larger cation Rb⁺, respectively. The observation that a single amino acid substitution can alter the monovalent cation specificity of thrombin from Na⁺ (Asp-189) to Li⁺ (Ser-189) or Rb⁺ (Glu-189) is unprecedented in the realm of monovalent cation-activated enzymes.

Received for publication, November 18, 2003 , and in revised form, December 12, 2003.

^* This work was supported in part by National Institutes of Health Grants HL49413, HL58141, and HL73813. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 314-362-4185; Fax: 314-747-5354; E-mail: enrico{at}biochem.wustl.edu.

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