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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.
| | The line below this paragraph, {{ABSTRACT_PUBMED_8855957}}, adds the Publication Abstract to the page |
| | (as it appears on PubMed at http://www.pubmed.gov), where 8855957 is the PubMed ID number. |
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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'' |
