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J. Biol. Chem., Vol. 280, Issue 21, 20824-20832, May 27, 2005

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Probing the Role of Asp-120(81) of Metallo--lactamase (IMP-1) by Site-directed Mutagenesis, Kinetic Studies, and X-ray Crystallography^*

Yoshihiro Yamaguchi, Takahiro Kuroki, Hisami Yasuzawa, Toshihiro Higashi, Wanchun Jin, Akiko Kawanami, Yuriko Yamagata¶, Yoshichika Arakawa||, Masafumi Goto, and Hiromasa Kurosaki

From the Departments of Structure-Function Physical Chemistry and ^¶Structural Biology, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-Honmachi, Kumamoto 862-0973 and the ^||Department of Bacterial Pathogenesis and Infection Control, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashi-Murayama, Tokyo 208-0011, Japan

Metallo--lactamase IMP-1 is a di-Zn(II) metalloenzyme that efficiently hydrolyzes -lactam antibiotics. Wild-type (WT) IMP-1 has a conserved Asp-120(81) in the active site, which plays an important role in catalysis. To probe the catalytic role of Asp-120(81) in IMP-1, the IMP-1 mutants, D120(81)A and D120(81)E, were prepared by site-directed mutagenesis, and various kinetics studies were conducted. The IMP-1 mutants exhibited 10²–10⁴-fold drops in k_cat values compared with WT despite the fact that they contained two Zn(II) ions in the active site. To evaluate the acid-base characteristics of Asp-120(81), the pH dependence for hydrolysis was examined by stopped-flow studies. No observable pK_a values between pH 5 and 9 were found for WT and D120(81)A. The rapid mixing of equimolar amounts of nitrocefin and all enzymes failed to result in the detection of an anion intermediate of nitrocefin at 650 nm. These results suggest that Asp-120(81) of IMP-1 is not a factor in decreasing the pK_a for the water bridging two Zn(II) ions and is not a proton donor to the anionic intermediate. In the case of D120(81)E, the nitrocefin hydrolysis product, which shows a maximum absorption at 460 nm, was bound to D120(81)E in the protonated form. The three-dimensional structures of D120(81)A and D120(81)E were also determined at 2.0 and 3.0 Å resolutions, respectively. In the case of D120(81)E, the Zn-Zn distance was increased by 0.3 Å compared with WT, due to the change in the coordination mode of Glu-120(81)OE1 and the positional shift in the conserved His-263(197) at the active site.

Received for publication, December 20, 2004 , and in revised form, March 15, 2005.

The atomic coordinates and structure factors (code 1WUO and 1WUP) 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 in part by H15-Shinkou-9 from the Ministry of Health Labor and Welfare of Japan and by a Grant-in-aid for Scientific Research 16390017 from Japan Society for the Promotion of Science. 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: Dept. of Structure-Function Physical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-Honmachi, Kumamoto 862-0973, Japan. Tel.: 81-96-371-4314; Fax: 81-96-371-4314; E-mail: yyamagu{at}gpo.kumamoto-u.ac.jp.

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