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| | {{STRUCTURE_1hde| PDB=1hde | SCENE= }} | | {{STRUCTURE_1hde| PDB=1hde | SCENE= }} |
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| - | '''HALOALKANE DEHALOGENASE MUTANT WITH PHE 172 REPLACED WITH TRP'''
| + | ===HALOALKANE DEHALOGENASE MUTANT WITH PHE 172 REPLACED WITH TRP=== |
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| - | ==Overview==
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| - | Conversion of halogenated aliphatics by haloalkane dehalogenase proceeds via the formation of a covalent alkyl-enzyme intermediate which is subsequently hydrolyzed by water. In the wild type enzyme, the slowest step for both 1,2-dichloroethane and 1,2-dibromoethane conversion is a unimolecular enzyme isomerization preceding rapid halide dissociation. Phenylalanine 172 is located in a helix-loop-helix structure that covers the active site cavity of the enzyme, interacts with the C1 beta of 1,2-dichloroethane during catalysis, and could be involved in stabilization of this helix-loop-helix region of the cap domain of the enzyme. To obtain more information about the role of this residue in dehalogenase function, we performed a mutational analysis of position 172 and studied the kinetics and X-ray structure of the Phe172Trp enzyme. The Phe172Trp mutant had a 10-fold higher Kcat/Km for 1-chlorohexane and a 2-fold higher Kcat for 1,2-dibromoethane than the wild-type enzyme. The X-ray structure of the Phe172Trp enzyme showed a local conformational change in the helix-loop-helix region that covers the active site. This could explain the elevated activity for 1-chlorohexane of the Phe172Trp enzyme, since it allows this large substrate to bind more easily in the active site cavity. Pre-steady-state kinetic analysis showed that the increase in Kcat found for 1,2-dibromoethane conversion could be attributed to an increase in the rate of an enzyme isomerization step that preceeds halide release. The observed conformational difference between the helix-loop-helix structures of the wild-type enzyme and the faster mutant suggests that the isomerization required for halide release could be a conformational change that takes place in this region of the cap domain of the dehalogenase. It is proposed that Phe172 is involved in stabilization of the helix-loop-helix structure that covers the active site of the enzyme and creates a rigid hydrophobic cavity for small apolar halogenated alkanes.
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| | + | {{ABSTRACT_PUBMED_8855957}} |
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| | ==About this Structure== | | ==About this Structure== |
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| | [[Category: Detoxification]] | | [[Category: Detoxification]] |
| | [[Category: Hydrolase]] | | [[Category: Hydrolase]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri May 2 18:43:40 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Tue Jul 1 08:01:40 2008'' |
Revision as of 05:01, 1 July 2008
Template:STRUCTURE 1hde
HALOALKANE DEHALOGENASE MUTANT WITH PHE 172 REPLACED WITH TRP
Template:ABSTRACT PUBMED 8855957
About this Structure
1HDE is a Single protein structure of sequence from Xanthobacter autotrophicus. Full crystallographic information is available from OCA.
Reference
Kinetic characterization and X-ray structure of a mutant of haloalkane dehalogenase with higher catalytic activity and modified substrate range., Schanstra JP, Ridder IS, Heimeriks GJ, Rink R, Poelarends GJ, Kalk KH, Dijkstra BW, Janssen DB, Biochemistry. 1996 Oct 8;35(40):13186-95. PMID:8855957
Page seeded by OCA on Tue Jul 1 08:01:40 2008
