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J. Biol. Chem., Vol. 279, Issue 14, 13962-13967, April 2, 2004

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The Functional Role of the Binuclear Metal Center in D-Aminoacylase

ONE-METAL ACTIVATION AND SECOND-METAL ATTENUATION^*

Wen-Lin Lai, Lien-Yang Chou, Chun-Yu Ting, Ralph Kirby¶, Ying-Chieh Tsai, Andrew H.-J. Wang||, and Shwu-Huey Liaw¶**

From the Structural Biology Program, Institute of Biochemistry, ^¶Faculty of Life Science, National Yang-Ming University, Taipei 11221, Taiwan, the ^||Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan, and the ^**Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei 11217, Taiwan

Our structural comparison of the TIM barrel metal-dependent hydrolase(-like) superfamily suggests a classification of their divergent active sites into four types: -binuclear, -mononuclear, -mononuclear, and metal-independent subsets. The D-aminoacylase from Alcaligenes faecalis DA1 belongs to the -mononuclear subset due to the fact that the catalytically essential Zn²⁺ is tightly bound at the site with coordination by Cys⁹⁶, His²²⁰, and His²⁵⁰, even though it possesses a binuclear active site with a weak binding site. Additional Zn²⁺, Cd²⁺, and Cu²⁺, but not Ni²⁺, Co²⁺, Mg²⁺, Mn²⁺, and Ca²⁺, can inhibit enzyme activity. Crystal structures of these metal derivatives show that Zn²⁺ and Cd²⁺ bind at the ₁ subsite ligated by His⁶⁷, His⁶⁹, and Asp³⁶⁶, while Cu²⁺ at the ₂ subsite is chelated by His⁶⁷, His⁶⁹ and Cys⁹⁶. Unexpectedly, the crystal structure of the inactive H220A mutant displays that the endogenous Zn²⁺ shifts to the ₃ subsite coordinated by His⁶⁷, His⁶⁹, Cys⁹⁶, and Asp³⁶⁶, revealing that elimination of the site changes the coordination geometry of the ion with an enhanced affinity. Kinetic studies of the metal ligand mutants such as C96D indicate the uniqueness of the unusual bridging cysteine and its involvement in catalysis. Therefore, the two metal-binding sites in the D-aminoacylase are interactive with partially mutual exclusion, thus resulting in widely different affinities for the activation/attenuation mechanism, in which the enzyme is activated by the metal ion at the site, but inhibited by the subsequent binding of the second ion at the site.

Received for publication, August 11, 2003 , and in revised form, January 20, 2004.

The atomic coordinates and structure factors (codes 1V51, 1RK6, 1RJP, 1V4Y, 1RJQ, 1RJR, and 1RK5 (see Table I)) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Science Council Grant NSC 92-2311-B-010-010. 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.

These authors contributed equally to the work.

To whom correspondence should be addressed: Dept. of Life Science, National Yang-Ming University, Taipei 11221, Taiwan. Tel.: 886-2-2826-7278; Fax: 886-2-2820-2449; E-mail: shliaw{at}ym.edu.tw.

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