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

4-Chlorobenzoyl-coenzyme A (4-CBA-COA) dehalogenase catalyzes the hydrolyti c dehalogenation of 4-CBA-CoA by attack of Asp145 on the C(4) of the substr ate benzoyl ring to form a Meisenheimer intermediate (EMc), followed by exp ulsion of chloride ion to form an arylated enzyme intermediate (EAr) and; f inally, ester hydrolysis in EAr to form 4-hydroxybenzoyl-CoA (4-HBA-CoA). T his study examines the contribution of the active site His90 to catalysis o f this reaction pathway: The His90 residue was replaced with glutamine by s ite-directed mutagenesis. X-ray crystallographic analysis of H90Q dehalogen ase complexed with 4-HBA-CoA revealed that the positions of the catalytic g roups are unchanged from those observed in the structure of the 4-HBA-CoA-w ild-type dehalogenase complex. The one exception is the Gln90 side chain, w hich is rotated away from the position of the His90 side chain. The vacated His90 site is occupied by two water molecules. Kinetic techniques were use d to evaluate ligand binding and catalytic turnover rates in the wild-type and H90Q mutant dehalogenases. The rate constants for 4-CBA-CoA (both 7 muM (-1) s(-1).) and 4-HBA-CoA (33 and 11 muM(-1) s(-1)) binding to the two deh alogenases are similar in value. For wild-type dehalogenase, the rate const ant for a single turnover is 2.3 s(-1) while that for multiple turnovers is 0.7 s(-1). For H90Q dehalogenase, these rate constants are 1.6 x 10(-2) an d 2 x 10(-4) s(-1). The rate constants for EMc formation in wild-type and m utant dehalogenase are similar to 200 s(-1) while the rate constants for EA r formation are 40 and 0.3 s(-1), respectively. The rate constant for hydro lysis of EAr in wild-type dehalogenase is 20 s(-1) and in the H90Q mutant, 0.13 s(-1). The 133-fold reduction in the rate of EAr formation in the muta nt may be the result of active site hydration, while the 154-fold reduction in the rate EAr hydrolysis may be the result of lost general base catalysi s. Substitution of the His90 with Gln also introduces a rate-limiting step which follows catalysis, and may involve renewing the catalytic site throug h a slow conformational change.