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J. Biol. Chem., Vol. 279, Issue 8, 7287-7295, February 20, 2004

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Structural and Enzymatic Analysis of Soybean -Amylase Mutants with Increased pH Optimum^*

Akira Hirata, Motoyasu Adachi, Atsushi Sekine, You-Na Kang, Shigeru Utsumi, and Bunzo Mikami

From the Laboratory of Food Quality Design and Development, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan

Comparison of the architecture around the active site of soybean -amylase and Bacillus cereus -amylase showed that the hydrogen bond networks (Glu³⁸⁰- (Lys²⁹⁵-Met⁵¹) and Glu³⁸⁰-Asn³⁴⁰-Glu¹⁷⁸) in soybean -amylase around the base catalytic residue, Glu³⁸⁰, seem to contribute to the lower pH optimum of soybean -amylase. To convert the pH optimum of soybean -amylase (pH 5.4) to that of the bacterial type enzyme (pH 6.7), three mutants of soybean -amylase, M51T, E178Y, and N340T, were constructed such that the hydrogen bond networks were removed by site-directed mutagenesis. The kinetic analysis showed that the pH optimum of all mutants shifted dramatically to a neutral pH (range, from 5.4 to 6.0-6.6). The K_m values of the mutants were almost the same as that of soybean -amylase except in the case of M51T, while the V_max values of all mutants were low compared with that of soybean -amylase. The crystal structure analysis of the wild type-maltose and mutant-maltose complexes showed that the direct hydrogen bond between Glu³⁸⁰ and Asn³⁴⁰ was completely disrupted in the mutants M51T, E178Y, and N340T. In the case of M51T, the hydrogen bond between Glu³⁸⁰ and Lys²⁹⁵ was also disrupted. These results indicated that the reduced pK_a value of Glu³⁸⁰ is stabilized by the hydrogen bond network and is responsible for the lower pH optimum of soybean -amylase compared with that of the bacterial -amylase.

Received for publication, August 25, 2003 , and in revised form, November 17, 2003.

The atomic coordinates and structure factors (code 1Q6C, 1Q6D, 1Q6E, 1Q6F, and 1Q6G) 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 a grant for the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. 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.: 81-774-38-3763; Fax: 81-774-38-3764; E-mail: mikami{at}kais.kyoto-u.ac.jp.

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