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KMID : 0578320100290050485
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Molecules and Cells
2010 Volume.29 No. 5 p.485 ~ p.492
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Molecular Characterization of a Novel Bacterial Aryl Acylamidase Belonging to the Amidase Signature Enzyme Family
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Ko Hyeok-Jin
Lee Eun-Woo
Lee Cheol-Koo
Kim Kyoung-Heon
Choi In-Geol
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Abstract
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In seeking aryl acylamidase (EC 3.5.1.13) acting on an amide bond in p-acetaminophenol (TylenolTM), we identified a novel gene encoding 496 residues of a protein. The gene revealed a conserved amidase signature region with a canonical catalytic triad. The gene was expressed in E. coli and characterized for its biochemical properties. The optimum pH and temperature for the activity on p-acetaminophenol were 10 and 37°C, respectively. The half-life of enzyme activity at 37°C was 192 h and 90% of its activity remained after 3 h incubation at 40°C. Divalent metals was found to inhibit the activity of enzyme. The Km values for various aryl acylamides such as 4-nitroacetanilide, p-acetaminophenol, phenacetin, 4-chloroacetanilide and acetanilide were 0.10, 0.32, 0.83, 1.9 and 19 mM, respectively. The reverse reaction activity (amide synthesis) was also examined using various chain lengths (C1~C4 and C10) of carboxylic donors and aniline as substrates. These kinetic parameters and substrate specificity in forward and reverse reaction indicated that the aryl acylamidase in this study has a preference for aryl substrate having polar functional groups and hydrophobic carboxylic donors.
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KEYWORD
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amidase signature enzymes, amide synthesis, aryl acylamidase, p-acetaminophenol
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