Kinetic characterization and X-ray structure of a mutant of haloalkane dehalogenase with higher catalytic activity and modified substrate range.
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.
Schanstra
J P
JP
Laboratory of Biochemistry, BIOSON Research Institute, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.
Ridder
I S
IS
Heimeriks
G J
GJ
Rink
R
R
Poelarends
G J
GJ
Kalk
K H
KH
Dijkstra
B W
BW
Janssen
D B
DB
D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D001419:Bacteria / Q000201:enzymology; D001965:Bromides / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D018360:Crystallography, X-Ray; D015946:Ethylene Dibromide / Q000378:metabolism; D018257:Helix-Loop-Helix Motifs; D006586:Hexanes / Q000378:metabolism; D006843:Hydrocarbons, Chlorinated / Q000378:metabolism; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D000596:Amino Acids; D001965:Bromides; D002712:Chlorides; D006586:Hexanes; D006843:Hydrocarbons, Chlorinated; D011994:Recombinant Proteins; D015946:Ethylene Dibromide; D010649:Phenylalanine; D006867:Hydrolases; C046137:haloalkane dehalogenase; C053283:1-chlorohexane