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University of Groningen Specificity and Kinetics of Haloalkane View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by University of Groningen University of Groningen Specificity and kinetics of haloalkane dehalogenase Schanstra, JP; Kingma, Jacob; Janssen, DB; Schanstra, Joost P. Published in: The Journal of Biological Chemistry DOI: 10.1074/jbc.271.25.14747 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 1996 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Schanstra, JP., Kingma, J., Janssen, DB., & Schanstra, J. P. (1996). Specificity and kinetics of haloalkane dehalogenase. The Journal of Biological Chemistry, 271(25), 14747-14753. https://doi.org/10.1074/jbc.271.25.14747 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 12-11-2019 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 25, Issue of June 21, pp. 14747–14753, 1996 © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Specificity and Kinetics of Haloalkane Dehalogenase* (Received for publication, January 18, 1996) Joost P. Schanstra, Jaap Kingma, and Dick B. Janssen‡ From the Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Haloalkane dehalogenase converts halogenated al- acting as a general base catalyst, leading to enzyme and bound kanes to their corresponding alcohols. The active site is products (EzROHzX). The final step is release of the products. buried inside the protein and lined with hydrophobic We recently found that halide release follows a complex path- residues. The reaction proceeds via a covalent sub- way and could limit the kcat (8). This reaction mechanism is strate-enzyme complex. This paper describes a steady- summarized in reaction scheme shown in Scheme I. state and pre-steady-state kinetic analysis of the conver- Besides the hydrophilic catalytic residues, the active site of sion of a number of substrates of the dehalogenase. The the dehalogenase is mainly lined with hydrophobic residues: 4 kinetic mechanism for the “natural” substrate 1,2-di- phenylalanines, 2 tryptophans, 2 leucines, a valine, and a pro- chloroethane and for the brominated analog and nema- line (4; Fig. 1). This hydrophobic environment and its relatively tocide 1,2-dibromoethane are given. In general, bromi- small size (37 Å3) predict a low affinity for polar and large nated substrates had a lower K , but a similar k than m cat compounds. The k values for 1,2-dichloroethane and 1,2- the chlorinated analogs. The rate of C-Br bond cleavage cat dibromoethane are similar, although the affinity for bromide was higher than the rate of C-Cl bond cleavage, which is ions is much higher than for chloride, and the carbon-bromine in agreement with the leaving group abilities of these bond is less stable than the carbon-chlorine bond. Thus, the halogens. The lower Km for brominated compounds therefore originates both from the higher rate of C-Br steady-state kinetics do not reflect the kinetics of carbon-halo- gen bond cleavage. bond cleavage and from a lower Ks for bromo-com- pounds. However, the rate-determining step in the con- To obtain further insight into the kinetics and specificity of the dehalogenase, we studied the steady-state kinetics of con- version (kcat) of 1,2-dibromoethane and 1,2-dichloroeth- ane was found to be release of the charged halide ion out version of a range of halogenated compounds, and the pre- of the active site cavity, explaining the different Km but steady-state kinetics of 1,2-dichloroethane and 1,2-dibromoeth- similar kcat values for these compounds. The study pro- ane conversion using stopped-flow fluorescence and rapid- vides a basis for the analysis of rate-determining steps quench-flow techniques. This paper presents a complete in the hydrolysis of various environmentally important description of the rates of the separate steps during 1,2-dibro- substrates. moethane and 1,2-dichloroethane conversion by haloalkane de- halogenase. The results show that in the dehalogenase reaction cleavage of C-Br bonds is faster than C-Cl bonds, in agreement Haloalkane dehalogenase is an enzyme capable of carbon- with the leaving group abilities of these halogens. The rate- halogen bond cleavage in xenobiotic halogenated aliphatic com- limiting step for 1,2-dichloroethane and 1,2-dibromoethane pounds. The enzyme converts a broad range of chlorinated conversion, however, is not carbon-halogen bond cleavage but (C2-C6) and brominated (C2-C8) alkanes to the corresponding release of the halide ion out of the active site cavity. alcohols and halides (1, 2). Some activity has also been ob- served with 2-bromoethanol, epichlorohydrin, and the EXPERIMENTAL PROCEDURES branched haloalkanes 1,2-dichloropropane and 1,2-dibro- Materials—Halogenated compounds were obtained from Janssen 2 mopropane (3). Although the enzyme is capable of performing Chimica (Beerse, Belgium) or from Merck (Darmstadt, Germany). H2O these dehalogenation reactions, which contribute to the detox- (99.8% v/v) was purchased from Merck or from Isotec Inc. (Miamisburg, ification of such compounds, the affinities and the turnover OH). Bacterial Strains and Plasmids—pGELAF1, an expression vector numbers with most substrates are low. based on pET-3d (9) with the dehalogenase gene (dhlA) under the Haloalkane dehalogenase is one of the few enzymes involved control of the T7-promoter and an additional f(1)1 origin for the pro- in degradation of xenobiotic compounds of which the x-ray duction of single-stranded DNA (10) was used to overexpress the de- structure is known (4). The reaction mechanism for 1,2-dichlo- halogenase in Escherichia coli strain BL21(DE3) (9). roethane has been solved using x-ray crystallography and site- Protein Expression and Purification—The enzyme was expressed directed mutagenesis studies (5–7). The reaction is initiated by and purified as described earlier (10). The buffers used during purifi- cation were TEMAG (10 mM Tris-sulfate, pH 7.5, 1 mM EDTA, 1 mM binding of the substrate into the Michaelis complex (EzRX), 2-mercaptoethanol, 3 mM sodium azide, and 10% (v/v) glycerol) and followed by nucleophilic attack of Asp-124 on the carbon atom PEMAG (10 mM sodium phosphate, pH 6.0, 1 mM EDTA, 1 mM 2-mer- to which the halogen is bound, leading to formation of an captoethanol, 3 mM sodium azide, and 10% (v/v) glycerol). The enzyme alkyl-enzyme intermediate (E-RzX). This covalent intermediate was concentrated with an Amicon ultrafiltration cell using a PM10 is subsequently hydrolyzed by activated water, with His-289 filter. Dehalogenase Assays and Protein Analysis—Dehalogenase assays were performed using colorimetric detection of halide release as de- * This work was supported by a grant from the Biotechnology Pro- scribed (1). Protein concentrations were determined with Coomassie gramme of the Dutch Ministry of Economic Affairs. The costs of publi- Brilliant Blue using bovine serum albumin as a standard. The concen- 4 cation of this article were defrayed in part by the payment of page tration of purified enzyme was determined using e280 5 4.87 3 10 charges. This article must therefore be hereby marked “advertisement” M21zcm21 calculated with the program DNASTAR (DNASTAR Inc., in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Madison, WI). The purified enzyme was analyzed with SDS-polyacryl- ‡ To whom correspondence should be addressed. Tel.: 31-50-3634208; amide gel electrophoresis, which showed that the purity of the prepa- Fax: 31-50-3634165, E-mail: [email protected]. rations was greater than 99% (data not shown). Solvent kinetic isotope 14747 14748 Specificity and Kinetics of Haloalkane Dehalogenase sets the lower limit for k1 (15). The fitting procedure was as follows. First, the pre-steady-state burst and single turnover data produced by rapid-quench experiments were simultaneously fit by numerical simu- SCHEME I lation of Scheme II by Gepasi, during which the quality of the fits was determined by visual inspection. The estimates of the rates derived 2 effects were determined with substrate dissolved in H2O. Assays were from the rapid-quench experiments were used as starting rates for 2 performed as described above using increasing concentrations of H2O. fitting of the fluorescence transients by the program FITSIM. This For the determination of the Km, alcohol or halide production was program combines numerical simulation of reaction schemes and non- measured in 4.5-ml incubations containing various concentrations of linear regression analysis (14). The rates derived in this step were then the substrate in 50 mM Tris-sulfate buffer, pH 8.2, and a suitable used to fit the rapid-quench-flow data. This cycle was repeated several amount of enzyme. Samples were incubated at 30 °C. The amount of times to determine the limits of the derived values and to converge to a enzyme was adjusted such that the amount of substrate converted in single solution.
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