Evidence for nucleophilic catalysis in the aromatic substitution reaction catalyzed by (4-chlorobenzoyl)coenzyme A dehalogenase.
(4-Chlorobenzoyl)coenzyme A dehalogenase catalyzes the hydrolytic dehalogenation of (4-chlorobenzoyl)coenzyme A (4-CBA-CoA) to (4-hydroxybenzoyl)coenzyme A (4-HBA-CoA). Rapid-quench techniques were used in conjunction with [14C]-4-CBA-CoA to test for the formation of a covalent enzyme intermediate during catalysis. The rate of [14C]-4-CBA-CoA (37 microM) consumption in the presence of a 2-fold excess of dehalogenase (75 microM) was determined to proceed at k = 6.5 s-1, coincident with the formation of an enzyme intermediate containing covalently bound radiolabel. The radiolabeled enzyme reached a maximum level at 100 ms, corresponding to 27% of the starting [14C]-4-CBA-CoA, before declining. The kinetics of formation and consumption of the radiolabeled enzyme observed during turnover are consistent with its intermediacy in the overall reaction. A single turnover reaction carried out in 98% 18O-enriched water produced 4-HBA-CoA with 73-75% 16O and 27-25% 18O at the benzoyl ring C(4)-OH. In contrast, a multiple turnover reaction carried out in 93% H2(18)O produced 4-HBA-CoA labeled at the C(4)-OH with 89% 18O and 11% 16O. These results were interpreted as evidence for formation of an aryl enzyme intermediate during 4-CBA-CoA hydrolytic dechlorination in the dehalogenase active site.
Yang
G
G
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
Liang
P H
PH
Dunaway-Mariano
D
D
D000214:Acyl Coenzyme A / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D006867:Hydrolases / Q000378:metabolism*; D007700:Kinetics; D010100:Oxygen / Q000378:metabolism; D010103:Oxygen Isotopes; D011549:Pseudomonas / Q000201:enzymology*; D014867:Water / Q000378:metabolism
D000214:Acyl Coenzyme A; D010103:Oxygen Isotopes; D014867:Water; C084334:4-chlorobenzoyl coenzyme A; D006867:Hydrolases; C084333:4-chlorobenzoyl coenzyme A dehalogenase; D010100:Oxygen