2009
DOI: 10.1002/cctc.200900041
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Suppressed Native Hydrolytic Activity of a Lipase to Reveal Promiscuous Michael Addition Activity in Water

Abstract: Suppression of the native hydrolytic activity of Pseudozyma antarctica lipase B (PalB) (formerly Candida antarctica lipase B) in water is demonstrated. By replacing the catalytic Ser 105 residue with an alanine unit, promiscuous Michael addition activity is favored. A Michael addition reaction between methyl acrylate and acetylacetone was explored as a model system. For the PalB Ser 105 Ala mutant, the hydrolytic activity was suppressed more than 1000 times and, at the same time, the Michael addition activity … Show more

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Cited by 48 publications
(27 citation statements)
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“…The M06-2X/MM energy of TS1 and TS2 relative (19.1 and 19.7 kcal·mol -1 , respectively) is very close to the free energy barrier that can be deduced from the experimental catalytic events measure by Berglund and co-workers (20.3 kcal·mol -1 ). 15 Our results suggest that the origin of the catalytic activity of the enzyme is due to favorable interactions established between the residues of the active site that create the oxyanion hole, Gln106 and Thr40, and the carbonyl oxygen atom of the substrate that develops a negative charge during the reaction and, in particular in the rate limiting TS. Nevertheless, there are other key residues for catalysis such as Ser105 that forms a covalent bond with the substrate stabilizing the intermediate states, or the Asp187 that is capable of modulating the pKa of His224, favoring its role as base or acid depending on the step of the process.…”
Section: Resultsmentioning
confidence: 74%
See 1 more Smart Citation
“…The M06-2X/MM energy of TS1 and TS2 relative (19.1 and 19.7 kcal·mol -1 , respectively) is very close to the free energy barrier that can be deduced from the experimental catalytic events measure by Berglund and co-workers (20.3 kcal·mol -1 ). 15 Our results suggest that the origin of the catalytic activity of the enzyme is due to favorable interactions established between the residues of the active site that create the oxyanion hole, Gln106 and Thr40, and the carbonyl oxygen atom of the substrate that develops a negative charge during the reaction and, in particular in the rate limiting TS. Nevertheless, there are other key residues for catalysis such as Ser105 that forms a covalent bond with the substrate stabilizing the intermediate states, or the Asp187 that is capable of modulating the pKa of His224, favoring its role as base or acid depending on the step of the process.…”
Section: Resultsmentioning
confidence: 74%
“…The M06-2X/MM energy of TS1 and TS2 relative to , which is the free energy barrier that can be deduced from the experimental catalytic events measure by Berglund and co-workers. 15 TS3, associated to the nucleophilic attack of the water molecule to the carbonyl carbon of the substrate, presents the highest energy barrier at this level of calculation. Nevertheless, it is important to note that energy of TS1 and TS2 are clearly higher than TS3.…”
Section: Resultsmentioning
confidence: 96%
“…1b shows the structure of CALB, which belongs to the large α/β hydrolase superfamily. [29] Initial experimental efforts with CALB wild type (wt) and S105A mutant (shown to have enhanced catalytic activity for carbon-carbon bond formation [20,22,23,24]) did not indicate any Diels-Alder activity.…”
Section: Fig 1: (A)mentioning
confidence: 99%
“…the ability of an enzyme to exhibit catalysis of other than its native reaction) towards e.g. aldol condensations, [19,20] epoxidations [21] and Michael additions, [22,23,24,25,26] not seldom enhanced by point mutations. [27] An often-seen feature in promiscuous catalysis is that only parts of the catalytic machinery are used, so that removal of one or more of For Production's Reference Only Click here to download Manuscript: Brinck_2010-DielsAlder_revised.pdf 2 …”
Section: Introductionmentioning
confidence: 99%
“…突变后水解能力降低了 1000 倍, 但是 Michael 加成的酶 活增加了 100 倍 [5] . 由这些推测可能因为丝氨酸通过与 His-Asp 形成氢键, 从而降低了 His-Asp 夺质子的能力, 结合于量子化学模拟, 确认反应是发生在酶的活性中 心, 但是这个结论仍需要更多的实验证明.…”
unclassified