Transient State Kinetic Analysis of the Chemical Intermediates Formed in the Enzymic Dehalogenation of 4-Chlorobenzoyl Coenzyme A

Liu, Rui‐Qin; Liang, Po‐Huang; Scholten, J. P.; Dunaway‐Mariano, Debra

doi:10.1021/ja00122a035

Cited by 27 publications

(34 citation statements)

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“…4). The formation of the tetrahedral addition intermediate and activation of the H 2 O molecule that effects the hydrolysis appears to have been decoupled into two separate steps by 4-CBD (V) [46,47]. Initial attack onto the 4-chlorobenzoyl-CoA substrate is by a suitably positioned aspartyl residue (Asp145), which reacts to form a covalently linked ester intermediate [11,48].…”

Section: -Chlorobenzoyl-coa Dehalogenase (4-cbd V)mentioning

confidence: 99%

Mechanisms and structures of crotonase superfamily enzymes – How nature controls enolate and oxyanion reactivity

Hamed

Batchelar

Clifton

et al. 2008

Cell. Mol. Life Sci.

111

145

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Structural and mechanistic studies on the crotonase superfamily (CS) are reviewed with the aim of illustrating how a conserved structural platform can enable catalysis of a very wide range of reactions. Many CS reactions have precedent in the 'carbonyl' chemistry of organic synthesis; they include alkene hydration/isomerization, aryl-halide dehalogenation, (de)carboxylation, CoA ester and peptide hydrolysis, fragmentation of beta-diketones and C-C bond formation, cleavage and oxidation. CS enzymes possess a canonical fold formed from repeated betabetaalpha units that assemble into two approximately perpendicular beta-sheets surrounded by alpha-helices. CS enzymes often, although not exclusively, oligomerize as trimers or dimers of trimers. Two conserved backbone NH groups in CS active sites form an oxyanion 'hole' that can stabilize enolate/oxyanion intermediates. The range and efficiency of known CS-catalyzed reactions coupled to their common structural platforms suggest that CS variants may have widespread utility in biocatalysis.

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Section: -Chlorobenzoyl-coa Dehalogenase (4-cbd V)mentioning

confidence: 99%

Mechanisms and structures of crotonase superfamily enzymes – How nature controls enolate and oxyanion reactivity

Hamed

Batchelar

Clifton

et al. 2008

Cell. Mol. Life Sci.

111

145

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“…We believe that this is because the rates of the two steps are better resolved in the H90Q-catalyzed reaction. Independent evidence for the intermediacy of EMc derives from a stoppedflow absorption kinetic analysis of the dehalogenase-catalyzed reactions of 4-CBA-CoA analogues (12). These findings, coupled with "chemical logic" (there is ample evidence in the literature for stepwise nucleophilic substitution on activated aromatic rings but none supporting a concerted mechanism) (24)(25)(26)(27), are most consistent with the two-covalent intermediate kinetic model of Scheme 2, and thus support its use in the determination of the microscopic rate constants.…”

Section: X-ray Crystal Structure Of the H90q Dehalogenase-4-hba-coa Cmentioning

confidence: 99%

Histidine 90 Function in 4-Chlorobenzoyl-Coenzyme A Dehalogenase Catalysis^,

Zhang

Wei²,

Luo³

et al. 2001

Biochemistry

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4-Chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydrolytic dehalogenation of 4-CBA-CoA by attack of Asp145 on the C(4) of the substrate benzoyl ring to form a Meisenheimer intermediate (EMc), followed by expulsion of chloride ion to form an arylated enzyme intermediate (EAr) and, finally, ester hydrolysis in EAr to form 4-hydroxybenzoyl-CoA (4-HBA-CoA). This study examines the contribution of the active site His90 to catalysis of this reaction pathway. The His90 residue was replaced with glutamine by site-directed mutagenesis. X-ray crystallographic analysis of H90Q dehalogenase complexed with 4-HBA-CoA revealed that the positions of the catalytic groups are unchanged from those observed in the structure of the 4-HBA-CoA-wild-type dehalogenase complex. The one exception is the Gln90 side chain, which is rotated away from the position of the His90 side chain. The vacated His90 site is occupied by two water molecules. Kinetic techniques were used to evaluate ligand binding and catalytic turnover rates in the wild-type and H90Q mutant dehalogenases. The rate constants for 4-CBA-CoA (both 7 µM -1 s -1 ) and 4-HBA-CoA (33 and 11 µM -1 s -1 ) binding to the two dehalogenases are similar in value. For wild-type dehalogenase, the rate constant for a single turnover is 2.3 s -1 while that for multiple turnovers is 0.7 s -1 . For H90Q dehalogenase, these rate constants are 1.6 × 10 -2 and 2 × 10 -4 s -1 . The rate constants for EMc formation in wild-type and mutant dehalogenase are ∼200 s -1 while the rate constants for EAr formation are 40 and 0.3 s -1 , respectively. The rate constant for hydrolysis of EAr in wild-type dehalogenase is 20 s -1 and in the H90Q mutant, 0.13 s -1 . The 133-fold reduction in the rate of EAr formation in the mutant may be the result of active site hydration, while the 154-fold reduction in the rate EAr hydrolysis may be the result of lost general base catalysis. Substitution of the His90 with Gln also introduces a rate-limiting step which follows catalysis, and may involve renewing the catalytic site through a slow conformational change.

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“…Following centrifugation to remove insoluble material, the protein was precipitated from the solution with the addition of solid ammonium sulfate to 70% saturation. The protein precipitate Scheme 1: Proposed Chemical Pathway of the 4-CBA-CoA Dehalogenase-Catalyzed Hydrolysis of 4-CBA-CoA to 4-HBA-CoA a a Liu et al (1995); Benning et al (1996); Yang et al (1996). FIGURE 1: Active-site region of wild-type 4-CBA-CoA dehalogenase derived from the crystal structure of the dehalogenase-4-HBACoA complex determined at 1.8 Å (Benning et al, 1996), showing (top) the arrangement of the Trp137, Asp145, and His90 side chains around the ligand hydroxybenzoyl ring and the positions of the Gly114 and Phe64 amide backbone NHs relative to the benzoyl CdO and (bottom) the arrangement of the Phe64, Phe82, Trp137, and Trp89 side chains around the ligand hydroxybenzoyl ring.…”

Section: Generalmentioning

confidence: 99%

Investigation of Substrate Activation by 4-Chlorobenzoyl-Coenzyme A Dehalogenase

et al. 1997

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4-Chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydrolysis of 4-CBACoA to 4-hydroxybenzoyl-coenzyme A (4-HBA-CoA), using the carboxylate side chain of aspartate 145 to displace the chloride from C(4) of the benzoyl ring. Previous UV-visible, Raman, and 13 C NMR studies of enzyme-bound substrate analog or product ligand indicated that the environment of the enzyme active site induces a significant reorganization of the benzoyl ring π-electrons. This observation was interpreted as evidence for electrophilic catalysis [Viz. active-site-induced polarization of electron density away from the ring C(4)] [

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Transient State Kinetic Analysis of the Chemical Intermediates Formed in the Enzymic Dehalogenation of 4-Chlorobenzoyl Coenzyme A

Cited by 27 publications

References 1 publication

Mechanisms and structures of crotonase superfamily enzymes – How nature controls enolate and oxyanion reactivity

Mechanisms and structures of crotonase superfamily enzymes – How nature controls enolate and oxyanion reactivity

Histidine 90 Function in 4-Chlorobenzoyl-Coenzyme A Dehalogenase Catalysis^,

Investigation of Substrate Activation by 4-Chlorobenzoyl-Coenzyme A Dehalogenase

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Transient State Kinetic Analysis of the Chemical Intermediates Formed in the Enzymic Dehalogenation of 4-Chlorobenzoyl Coenzyme A

Cited by 27 publications

References 1 publication

Mechanisms and structures of crotonase superfamily enzymes – How nature controls enolate and oxyanion reactivity

Mechanisms and structures of crotonase superfamily enzymes – How nature controls enolate and oxyanion reactivity

Histidine 90 Function in 4-Chlorobenzoyl-Coenzyme A Dehalogenase Catalysis,

Investigation of Substrate Activation by 4-Chlorobenzoyl-Coenzyme A Dehalogenase

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Histidine 90 Function in 4-Chlorobenzoyl-Coenzyme A Dehalogenase Catalysis^,