Escherichia coli TAP (thioesterase I, EC 3.1.2.2) is a multifunctional enzyme with thioesterase, esterase, arylesterase, protease and lysophospholipase activities. Previous crystal structural analyses identified its essential amino acid residues as those that form a catalytic triad (Ser10-Asp154-His157) and those involved in forming an oxyanion hole (Ser10-Gly44-Asn73). To gain an insight into the biochemical roles of each residue, site-directed mutagenesis was employed to mutate these residues to alanine, and enzyme kinetic studies were conducted using esterase, thioesterase and amino-acid-derived substrates. Of the residues, His157 is the most important, as it plays a vital role in the catalytic triad, and may also play a role in stabilizing oxyanion conformation. Ser10 also plays a very important role, although the small residual activity of the S10A variant suggests that a water molecule may act as a poor substitute. The water molecule could possibly be endowed with the nucleophilic-attacking character by His157 hydrogen-bonding. Asp154 is not as essential compared with the other two residues in the triad. It is close to the entrance of the substrate tunnel, therefore it predominantly affects substrate accessibility. Gly44 plays a role in stabilizing the oxyanion intermediate and additionally in acyl-enzyme-intermediate transformation. N73A had the highest residual enzyme activity among all the mutants, which indicates that Asn73 is not as essential as the other mutated residues. The role of Asn73 is proposed to be involved in a loop75–80 switch-move motion, which is essential for the accommodation of substrates with longer acyl-chain lengths.
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Research Article|
June 14 2006
Functional role of catalytic triad and oxyanion hole-forming residues on enzyme activity of Escherichia coli thioesterase I/protease I/phospholipase L1
Li-Chiun Lee;
Li-Chiun Lee
*Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, 20224, Taiwan
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Ya-Lin Lee;
Ya-Lin Lee
1
†Department of Nutrition and Food Sciences, Fu-Jen Catholic University, Taipei County, 24205, Taiwan
1Correspondence may be addressed to either J.-F.S. (email presid@dragon.nchu.edu.tw) or Y.-L.L. (email nutr2032@mails.fju.edu.tw).
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Ruey-Jyh Leu;
Ruey-Jyh Leu
‡Institute of Botany, Academia Sinica, Nankang, Taipei, 11529, Taiwan
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Jei-Fu Shaw
Jei-Fu Shaw
1
*Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, 20224, Taiwan
‡Institute of Botany, Academia Sinica, Nankang, Taipei, 11529, Taiwan
§Department of Food Science and Biotechnology, National Chung Hsing University, Taichung, 40227, Taiwan
1Correspondence may be addressed to either J.-F.S. (email presid@dragon.nchu.edu.tw) or Y.-L.L. (email nutr2032@mails.fju.edu.tw).
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Publisher: Portland Press Ltd
Received:
October 10 2005
Revision Received:
February 27 2006
Accepted:
March 06 2006
Accepted Manuscript online:
March 06 2006
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2006
Biochem J (2006) 397 (1): 69–76.
Article history
Received:
October 10 2005
Revision Received:
February 27 2006
Accepted:
March 06 2006
Accepted Manuscript online:
March 06 2006
Citation
Li-Chiun Lee, Ya-Lin Lee, Ruey-Jyh Leu, Jei-Fu Shaw; Functional role of catalytic triad and oxyanion hole-forming residues on enzyme activity of Escherichia coli thioesterase I/protease I/phospholipase L1. Biochem J 1 July 2006; 397 (1): 69–76. doi: https://doi.org/10.1042/BJ20051645
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