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Role of active site binding interactions in 4-chlorobenzoyl-coenzyme A dehalogenase catalysis
1Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131, USA.
Insights
This study reveals how specific binding interactions enhance 4-Chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalysis. Key hydrogen bonds and CoA nucleotide interactions significantly lower the energy barrier, increasing catalytic efficiency by 10^6-fold.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Structural biology
Background:
- 4-Chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase facilitates hydrolytic dehalogenation through a complex mechanism.
- Understanding enzyme-substrate binding is crucial for elucidating catalytic strategies.
Purpose of the Study:
- To investigate the role of specific binding interactions in the catalytic mechanism of 4-CBA-CoA dehalogenase.
- To quantify the contribution of these interactions to catalysis.
Main Methods:
- Site-directed mutagenesis and chemical synthesis were used to modify enzyme and substrate groups.
- Changes in Gibbs free energy (DeltaDeltaG(ES) and DeltaDeltaG) were measured to assess binding contributions.
- Analysis focused on cases where DeltaDeltaG > DeltaDeltaG(ES) to identify catalytically relevant interactions.
Main Results:
- Hydrogen bonds between Gly114/Phe64 and the substrate's C=O group stabilize the transition state by 3.1 kcal/mol.
- Enzyme-substrate CoA nucleotide moiety interactions stabilize the transition state by 3.3 kcal/mol.
- These binding interactions collectively enhance catalytic efficiency (k(cat)/K(m)) by approximately 10^6-fold.
Conclusions:
- Specific binding interactions are critical for the catalytic power of 4-CBA-CoA dehalogenase.
- Stabilization of the transition state through these interactions significantly lowers the activation energy.
- The findings provide insights into enzyme design and catalytic mechanisms.
Abstract:
4-Chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydrolytic dehalogenation of 4-CBA-CoA to 4-hydroxybenzoyl-CoA (4-HBA-CoA) via a multistep mechanism involving initial attack of Asp145 on C(4) of the substrate benzoyl ring to form a Meisenheimer intermediate (EMc), followed by expulsion of the chloride ion to form an arylated enzyme intermediate (EAr) and then ester hydrolysis in the EAr to form product. This study examines the role of binding interactions in dehalogenase catalysis. The enzyme and substrate groups positioned for favorable binding interaction were identified from the X-ray crystal structure of the enzyme-4-HBA-3'-dephospho-CoA complex. These groups were individually modified (via site-directed mutagenesis or chemical synthesis) for the purpose of disrupting the binding interaction. The changes in the Gibbs free energy of the enzyme-substrate complex (DeltaDeltaG(ES)) and enzyme-transition state complex (DeltaDeltaG) brought about by the modification were measured. Cases where DeltaDeltaG exceeds DeltaDeltaG(ES) are indicative of binding interactions used for catalysis. On the basis of this analysis, we show that the H-bond interactions between the Gly114 and Phe64 backbone amide NHs and the substrate benzoyl C=O group contribute an additional 3.1 kcal/mol of stabilization at the rate-limiting transition state. The binding interactions between the enzyme and the substrate CoA nucleotide moiety also intensify in the rate-limiting transition state, reducing the energy barrier to catalysis by an additional 3.3 kcal/mol. Together, these binding interactions contribute approximately 10(6) to the k(cat)/K(m).