Kinetic Characterization and X-ray Structure of a Mutant of Haloalkane Dehalogenase with Higher Catalytic Activity and Modified Substrate Range^,

Abstract

Kinetic characterization and X-ray structure of a mutant of haloalkane dehalogenase with higher catalytic activity and modified substrate range Schanstra, Joost P.; Ridder, Ivo S.; Heimeriks, Gaston J.; Rink, Rick; Poelarends, Gerrit; Kalk, Kor H.; Dijkstra, Bauke W.; Janssen, Dick 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 VersionPublisher'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, J. P., Ridder, I. S., Heimeriks, G. J., Rink, R., Poelarends, G. J., Kalk, K. H., ... Janssen, D. B. (1996). Kinetic characterization and X-ray structure of a mutant of haloalkane dehalogenase with higher catalytic activity and modified substrate range. Biochemistry, 35(40), 13186-13195. DOI: 10.1021/bi961151a 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. ABSTRACT: 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 helixloop-helix structure that covers the active site cavity of the enzyme, interacts with the Cl 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 k cat /K m for 1-chlorohexane and a 2-fold higher k cat 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 k cat found for 1,2-dibromoethane conversion could be attributed to an increase in the...

Catalytic Mechanism of the Haloalkane Dehalogenase LinB from Sphingomonas paucimobilis UT26

“…Moreover, site-directed mutagenesis studies of DhlA demonstrated that even a single point mutation may result in the change of the rate-limiting step. Hydrolysis of the alkyl-enzyme intermediate and not a halide release was the slowest reaction step in three single point mutants of DhlA: V226A, F172W, and W175Y (22)(23)(24).…”

Comparison of formation of reactive conformers for the S N 2 displacements by CH 3 CO\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\setlength{\oddsidemargin}{-69pt}\begin{document}\begin{equation*}{\mathrm{_{2}^{-}}}\end{equation*}\end{document} in water and by Asp124-CO\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wa

“…In summary, the free energy difference between the NAC and the ground state of AcO Ϫ and DCE at 1 M concentration is 3.8 kcal͞mol, whereas the free energy difference between DhlA⅐DCE and DhlA⅐NAC is 1.2 kcal͞mol. The activation energy is 26 kcal͞mol in water (2) and 15.3 kcal͞mol at the enzyme active site (1). Thus, the free energy difference in formation of NAC (2.6 kcal͞mol) accounts for Ϸ25% of the advantage of the enzymatic reaction (⌬⌬G ϭ 11 kcal͞mol).…”

“…This reaction can be compared with the reaction in water with CH 3 CO 2 Ϫ (AcO Ϫ ) as a nucleophile (Scheme 1). The activation barrier (⌬G ) for the displacement of Cl Ϫ from dichloroethane (DCE) is 15.3 kcal͞mol for the enzymatic reaction (1) and Ϸ26 kcal͞mol for the nonenzymatic counterpart in water (2). The first goal of this study is to devise means of determining the time-dependent mechanism of forming contact pairs from two separate reactants in water that satisfy the structural restraints of a near attack conformation (NAC).…”

Haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 refined at 1.15 Å resolution

“…As several of its substrates are toxic xenobiotics, the enzyme is of fundamental interest in environmental biotechnology (Stucki & Thu È er, 1994. Detailed biochemical and crystallographic studies have supplied insight into the structure and catalytic mechanism of DhlA (Franken et al, 1991;Krooshof et al, 1998;Pries et al, 1994;Schanstra, Kingma et al, 1996;Schanstra, Ridder et al, 1996;Verschueren, Franken et al, 1993;Verschueren, Selje  e et al, 1993). The protein consists of two domains.…”