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J. Biol. Chem., Vol. 281, Issue 52, 40057-40064, December 29, 2006

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Arg-158 Is Critical in Both Binding the Substrate and Stabilizing the Transition-state Oxyanion for the Enzymatic Reaction of Malonamidase E2^*

Young Sung Yun, Wook Lee, Sejeong Shin, Byung-Ha Oh, and Kwan Yong Choi¹

From the Department of Life Sciences, National CRI Center for Biomolecular Recognition, Pohang University of Science and Technology, Pohang 790-784, South Korea

Malonamidase E2 (MAE2) from Bradyrhizobium japonicum is an enzyme that hydrolyzes malonamate to malonate and has a Ser-cis-Ser-Lys catalytic triad at the active site. The crystal structures of wild type and mutant MAE2 exhibited that the guanido group of Arg-158 could be involved in the binding of malonamate in which the negative charge of the carboxyl group could destabilize a negatively charged transition-state oxyanion in the enzymatic reaction. In an attempt to elucidate the specific roles of Arg-158, site-directed mutants, R158Q, R158E, and R158K, were prepared (see Table 1). The crystal structure of R158Q determined at 2.2 Å resolution showed that the guanido group of Arg-158 was important for the substrate binding with the marginal structural change upon the mutation. The k_cat value of R158Q significantly decreased by over 1500-fold and the catalytic activity of R158E could not be detected. The k_cat value of R158K was similar to that of the wild type with the K_m value drastically increased by 100-fold, suggesting that Lys-158 of R158K can stabilize the negative charge of the carboxylate in the substrate to some extent and contribute to the stabilization of the transition-state oxyanion, but a single amine group of Lys-158 in R158K could not precisely anchor the carboxyl group of malonamate compared with the guanido group of Arg-158. Our kinetic and structural evidences demonstrate that Arg-158 in MAE2 should be critical to both binding the substrate and stabilizing the transition-state oxyanion for the catalytic reaction of MAE2.

Received for publication, May 11, 2006 , and in revised form, October 4, 2006.

The atomic coordinates and structure factors (code 1OBK) 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 the Korea Research Foundation Grant (KRF-2004-005-C00004) and in part by the Brain Korea 21 project (to W. L.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ To whom correspondence should be addressed: Program of Biomolecular Function, Dept. of Life Sciences, Pohang University of Science and Technology, Pohang 790-784, South Korea. Tel.: 82-54-279-2295; Fax: 82-54-279-2199; E-mail: kchoi{at}postech.ac.kr.

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