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)
Taylor
K L
KL
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742, USA.
Liu
R Q
RQ
Liang
P H
PH
Price
J
J
Dunaway-Mariano
D
D
Tonge
P J
PJ
Clarkson
J
J
Carey
P R
PR
D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism*; D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D004746:Enoyl-CoA Hydratase / Q000737:chemistry / Q000378:metabolism; D006867:Hydrolases / Q000737:chemistry / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D011549:Pseudomonas / Q000201:enzymology*; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D013251:Steroid Isomerases / Q000737:chemistry / Q000378:metabolism
D000214:Acyl Coenzyme A; C084334:4-chlorobenzoyl coenzyme A; D000426:Alcohol Dehydrogenase; D006867:Hydrolases; C084333:4-chlorobenzoyl coenzyme A dehalogenase; D004746:Enoyl-CoA Hydratase; D013251:Steroid Isomerases; C022660:steroid delta-isomerase