Histidine 289 is essential for hydrolysis of the alkyl-enzyme intermediate of haloalkane dehalogenase.
Haloalkane dehalogenase (DhlA) from Xanthobacter autotrophicus GJ10 catalyzes the hydrolytic cleavage of carbon-halogen bonds in a broad range of halogenated aliphatic compounds. Previous work has shown that Asp124, which is located close to the internal substrate-binding cavity, carries out a nucleophilic attack on the C-alpha of the alkylhalide, displacing the halogen. The resulting alkyl-enzyme intermediate is subsequently hydrolyzed. In order to study the role of His289 in the hydrolysis of the intermediate, a His289-->Gln mutant was constructed by site-directed mutagenesis. The purified mutant enzyme was not catalytically active with haloalkanes, but a halide burst stoichiometric to the amount of enzyme was observed with 1,2-dibromoethane. Using ion spray mass spectrometry, accumulation of the covalent alkyl-enzyme and binding of the alkyl moiety of the substrate to an Asp124-containing tryptic peptide were shown. Fluorescence-quenching experiments indicated that halide ions are strongly bound by the alkyl-enzyme but not by the substrate-free enzyme. The results show that His289 is the base catalyst for the dealkylation of the covalent intermediate, but that it is not essential for the initial nucleophilic attack of Asp124 on the C-1 atom of the haloalkane. Furthermore, the halide ion that is released in the first step probably leaves the active site only after hydrolysis of the alkyl-enzyme.
Pries
F
F
Department of Biochemistry, University of Groningen, The Netherlands.
Kingma
J
J
Krooshof
G H
GH
Jeronimus-Stratingh
C M
CM
Bruins
A P
AP
Janssen
D B
DB
D000478:Alkylation; D001426:Bacterial Proteins / Q000737:chemistry; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers / Q000737:chemistry; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology*; D006639:Histidine / Q000737:chemistry; D006867:Hydrolases / Q000737:chemistry*; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000032:analysis; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D001426:Bacterial Proteins; D017931:DNA Primers; D010446:Peptide Fragments; D006639:Histidine; D006867:Hydrolases; C046137:haloalkane dehalogenase