Replacement of Tryptophan Residues in Haloalkane Dehalogenase Reduces Halide Binding and Catalytic Activity

Kennes, Christian; Pries, Frens; Krooshof, Geja H.; Bokma, Evert; Kingma, Jaap; Janssen, Dick B.

doi:10.1111/j.1432-1033.1995.tb20277.x

Cited by 12 publications

(20 citation statements)

References 24 publications

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“…Mutagenesis of active site residues Trp175 and Asp260 of DhlA, and subsequent structural and kinetic analysis, corroborated the above observations. These experiments showed that halide release in these mutants was accelerated and not limiting maximum turnover as was found for wild-type DhlA (34,35).…”

Section: Transient-state Kinetic Analysis Of 13-dibromopropane Convesupporting

confidence: 57%

Steady-State and Pre-Steady-State Kinetic Analysis of Halopropane Conversion by a Rhodococcus Haloalkane Dehalogenase

et al. 2003

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Section: Transient-state Kinetic Analysis Of 13-dibromopropane Convesupporting

confidence: 57%

Steady-State and Pre-Steady-State Kinetic Analysis of Halopropane Conversion by a Rhodococcus Haloalkane Dehalogenase

et al. 2003

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“…The energy difference of the reactants is lower by 2.1 kcal͞mol in the enzyme relative to the gas phase. The amount of stabilization energy provided by the enzyme is not too surprising given the results of mutational studies of Trp-125 and Trp-175 (5,29). Single mutations of these two tryptophans to glutamines showed that the catalytic efficiency (k cat ͞K m ) of DhlA is Ϸ100-fold lower for W175Q relative to W125Q for DCE.…”

Section: Resultsmentioning

confidence: 99%

The importance of reactant positioning in enzyme catalysis: A hybrid quantum mechanics/molecular mechanics study of a haloalkane dehalogenase

Lau

Kahn

Bash

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

102

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Hybrid quantum mechanics͞molecular mechanics calculations using Austin Model 1 system-specific parameters were performed to study the S N2 displacement reaction of chloride from 1,2-dichloroethane (DCE) by nucleophilic attack of the carboxylate of acetate in the gas phase and by Asp-124 in the active site of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10. The activation barrier for nucleophilic attack of acetate on DCE depends greatly on the reactants having a geometry resembling that in the enzyme or an optimized gas-phase structure. It was found in the gas-phase calculations that the activation barrier is 9 kcal͞mol lower when dihedral constraints are used to restrict the carboxylate nucleophile geometry to that in the enzyme relative to the geometries for the reactants without dihedral constraints. The calculated quantum mechanics͞molecular mechanics activation barriers for the enzymatic reaction are 16.2 and 19.4 kcal͞mol when the geometry of the reactants is in a near attack conformer from molecular dynamics and in a conformer similar to the crystal structure (DCE is gauche), respectively. This haloalkane dehalogenase lowers the activation barrier for dehalogenation of DCE by 2-4 kcal͞mol relative to the single point energies of the enzyme's quantum mechanics atoms in the gas phase. S N2 displacements of this sort in water are infinitely slower than in the gas phase. The modest lowering of the activation barrier by the enzyme relative to the reaction in the gas phase is consistent with mutation experiments.H aloalkane dehalogenases catalyze the hydrolytic cleavage of carbon-halogen bonds in aliphatic and aromatic halogenated compounds. The haloalkane dehalogenase from the nitrogen-fixing hydrogen bacterium Xanthobacter autotrophicus GJ10 (DhlA) prefers 1,2-dichloroethane (DCE) as substrate and converts it to 2-chloroethanol and chloride (1). A catalytic triad consisting of Asp-124, His-289, and Asp-260 is the central residue in the dehalogenation reaction (Scheme 1). On binding DCE in the predominantly hydrophobic active site, it undergoes S N 2 displacement of chloride by nucleophilic attack of Asp-124-COO Ϫ to form an ester intermediate at the rate of 50 Ϯ 10 s Ϫ1(1, 2). The ester intermediate is subsequently hydrolyzed by an activated water molecule. The dyad of His-289 and Asp-260 is thought to be responsible for activating the water molecule (3). This enzyme functions most efficiently at pH 8.2, likely because the imidazole NE2 of His-289 needs to be unprotonated for the hydrolysis reaction to proceed. Dehalogenases are of great interest because they are able to react with halogenated molecules under mild conditions (4). Many halogenated molecules are pollutants, and bioremediation is a highly desirable method for removing these harmful molecules from the environment. Dehalogenases have not existed in nature for a long time andhave not yet evolved into optimal enzymes. For example, the catalytic efficiency for DhlA is 4,550 M Ϫ1 ⅐s Ϫ1 for DCE (5), as compared with 5.6 ϫ 10 7 M Ϫ1 ⅐s Ϫ1for...

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“…Mutations in the residues of the catalytic triad result in loss of activity (Pries et al 1995a;Pries et al 1995b;Hynkova et al 1999), whereas mutations in the halide-stabilizing residues result in significantly reduced activity (Kennes et al 1995;Schindler et al 1999). The residues of the catalytic triad are directly involved in the dehalogenation reaction and their lower flexibility is expected to be favorable for the catalysis.…”

Section: Discussionmentioning

confidence: 99%

Functionally relevant motions of haloalkane dehalogenases occur in the specificity-modulating cap domains

Otyepka¹

2002

Protein Science

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One-nanosecond molecular dynamics trajectories of three haloalkane dehalogenases (DhlA, LinB, and DhaA) are compared. The main domain was rigid in all three dehalogenases, whereas the substrate specificity-modulating cap domains showed considerably higher mobility. The functionally relevant motions were spread over the entire cap domain in DhlA, whereas they were more localized in LinB and DhaA. The highest amplitude of essential motions of DhlA was noted in the ␣4Ј-helix-loop-␣4-helix region, formerly proposed to participate in the large conformation change needed for product release. The highest amplitude of essential motions of LinB and DhaA was observed in the random coil before helix 4, linking two domains of these proteins. This flexibility is the consequence of the modular composition of haloalkane dehalogenases. Two members of the catalytic triad, that is, the nucleophile and the base, showed a very high level of rigidity in all three dehalogenases. This rigidity is essential for their function. One of the halide-stabilizing residues, important for the catalysis, shows significantly higher flexibility in DhlA compared with LinB and DhaA. Enhanced flexibility may be required for destabilization of the electrostatic interactions during the release of the halide ion from the deeply buried active site of DhlA. The exchange of water molecules between the enzyme active site and bulk solvent was very different among the three dehalogenases. The differences could be related to the flexibility of the cap domains and to the number of entrance tunnels.

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Replacement of Tryptophan Residues in Haloalkane Dehalogenase Reduces Halide Binding and Catalytic Activity

Cited by 12 publications

References 24 publications

Steady-State and Pre-Steady-State Kinetic Analysis of Halopropane Conversion by a Rhodococcus Haloalkane Dehalogenase

Steady-State and Pre-Steady-State Kinetic Analysis of Halopropane Conversion by a Rhodococcus Haloalkane Dehalogenase

The importance of reactant positioning in enzyme catalysis: A hybrid quantum mechanics/molecular mechanics study of a haloalkane dehalogenase

Functionally relevant motions of haloalkane dehalogenases occur in the specificity-modulating cap domains

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