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Histidine 90 function in 4-chlorobenzoyl-coenzyme a dehalogenase catalysis.

W Zhang1, Y Wei, L Luo

  • 1Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131, USA.

Biochemistry
|November 7, 2001
PubMed
Summary

The study investigated the role of His90 in 4-chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase activity. Replacing His90 with glutamine significantly reduced catalytic rates, suggesting His90 is crucial for general base catalysis and efficient dehalogenation.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Structural Biology

Background:

  • 4-chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase facilitates hydrolytic dehalogenation.
  • The catalytic mechanism involves a Meisenheimer intermediate (EMc) and an arylated enzyme intermediate (EAr).
  • The role of active site His90 in this pathway requires elucidation.

Purpose of the Study:

  • To investigate the catalytic contribution of His90 in 4-CBA-CoA dehalogenase.
  • To characterize the impact of His90 substitution on the enzyme's reaction pathway and kinetics.

Main Methods:

  • Site-directed mutagenesis was employed to replace His90 with glutamine (H90Q mutant).
  • X-ray crystallography was used to determine the structure of the H90Q mutant complexed with 4-hydroxybenzoyl-CoA (4-HBA-CoA).

Related Experiment Videos

  • Kinetic techniques were applied to assess ligand binding and catalytic turnover rates for both wild-type and mutant enzymes.

    • Main Results:

    • X-ray crystallography revealed no significant changes in catalytic group positions in the H90Q mutant, except for the Gln90 side chain rotation and water molecule occupation.
    • Ligand binding rates for 4-CBA-CoA and 4-HBA-CoA were similar between wild-type and H90Q dehalogenases.
    • The H90Q mutation drastically reduced catalytic turnover rates, with a 133-fold decrease in EAr formation and a 154-fold decrease in EAr hydrolysis rates.

    Conclusions:

    • His90 is essential for efficient catalysis, likely acting as a general base catalyst for ester hydrolysis.
    • Active site hydration and a potential slow conformational change may contribute to the reduced catalytic efficiency in the H90Q mutant.
    • The findings highlight the critical role of His90 in the dehalogenation mechanism of 4-CBA-CoA dehalogenase.