Evidence for electrophilic catalysis in the 4-chlorobenzoyl-CoA dehalogenase reaction: UV, Raman, and 13C-NMR spectral studies of dehalogenase complexes of benzoyl-CoA adducts.

This paper reports on the mechanism of substrate activation by the enzyme 4-chlorobenzoyl coenzyme A dehalogenase. This enzyme catalyzes the hydrolytic dehalogenation of 4-chlorobenzoyl coenzyme A (4-CBA-CoA) to form 4-hydroxybenzoyl coenzyme A (4-HBA-CoA). The mechanism of this reaction is known to involve attack of an active site carboxylate (Asp or Glu side chain) at C(4) of the substrate benzoyl ring to form a Meisenheimer complex. Loss of chloride ion from this intermediate results in the formation of an arylated enzyme intermediate. The arylated enzyme is hydrolyzed to free enzyme plus 4-HBA-CoA by the addition of water at the acyl carbon [Yang, G., Liang, P.-H., & Dunaway-Mariano, D. (1994) Biochemistry 33, 8527]. The present studies have focused on the activation of the 4-CBA-CoA for nucleophilic attack by the active site carboxylate group. UV-visible, 13C-NMR, and Raman spectroscopic techniques were used to monitor changes in the distribution of the pi electrons of the benzoyl moiety of benzoyl-CoA adducts [substituted at C(4) with methyl (4-MeBA-CoA), methoxy (4-MeOBA-CoA), or hydroxyl (4-HBA-CoA) groups or at C(2) or C(3) with a hydroxyl group (2-HBA-CoA and 3-HBA-CoA)] resulting from the binding of these ligands to the dehalogenase active site. The UV-visible spectra measured for 4-HBA-CoA in aqueous buffer at pH 7.5 and in the dehalogenase active site revealed that a large red shift (from 292 to 373 nm) in the lambda max of the benzoyl moiety occurs upon binding.(ABSTRACT TRUNCATED AT 250 WORDS)