Mutational and Structural Studies of the Diisopropylfluorophosphatase from Loligo vulgaris Shed New Light on the Catalytic Mechanism of the Enzyme
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- 作者：Vicky Katsemi ; Christian Lü ; cke ; Juergen Koepke ; Frank Lö ; hr ; Steffen Maurer ; Gü ; nter Fritzsch ; and Heinz Rü ; terjans
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：June 28, 2005
- 年：2005
- 卷：44
- 期：25
- 页码：9022 - 9033
- 全文大小：431K
- 年卷期：v.44,no.25(June 28, 2005)
- ISSN：1520-4995
文摘
The active site, the substrate binding site, and the metal binding sites of the diisopropylfluorophosphatase (DFPase) from Loligo vulgaris have been modified by means of site-directed mutagenesisto improve our understanding of the reaction mechanism. Enzymatic characterization of mutants locatedin the major groove of the substrate binding pocket indicates that large hydrophobic side chains at thesepositions are favorable for substrate turnover. Moreover, the active site residue His287 proved to bebeneficial, but not essential, for DFP hydrolysis. In most cases, hydrophobic side chains at position 287led to significant catalytic activities although reduced relative to the wild-type enzyme. With respect tothe Ca-1 binding site, where catalysis occurs, various mutants indicated that the net charge at this calcium-binding site as well as the relative positions of the charged calcium ligands is crucial for catalytic activity.The importance of the electrostatic potential at the active site was furthermore revealed by various mutationsof residues lining the interior of the central water-filled tunnel, which traverses the entire protein structure.In this respect, the structural features of residue His181, which is located at the opposite end of the DFPasetunnel relative to the active site, were characterized extensively. It was concluded that a tunnel-spanninghydrogen bond network, which includes a large number of apparently slow exchanging water molecules,relays any modifications in the electrostatics of the system to the active site, thus affecting the catalyticreactivity of the enzyme.