Dissecting and expanding catalysis in 4-chlorobenzoyl-CoA dehalogenase.

摘要

4-Chlorobenzoyl-Coenzyme A (4-CBA-CoA) dehalogenase isolated from Pseudomonas sp. strain CBS3 catalyzes a novel hydrolytic dehalogenation of 4-CBA-CoA to 4-Hydroxybenzoyl-CoA (4-HBA-CoA). This thesis examines the contributions of active site residues to substrate binding, product release, and catalysis. The rational engineering of the dehalogenase with enhanced activity towards 4-Fluorobenzoy-CoA (4FBA-CoA) and 4-Nitrobenzoyl-CoA (4-NBA-CoA) is also examined.;The binding interactions between enzyme and substrate/product ligand were explored. The results showed that (i) substrate binding is fast and conforms to a lock-and-key model, (ii) product release is partially rate limiting, (iii) slow product release is due to binding interaction between the CoA phosphoryl groups and three Arginine residues, and to the sliding of the hydrophobic benzoyl-ring out of a tightly packed hydrophobic pocket.;The microscopic rate constants of reaction pathway were defined by using transient kinetic study. The results indicated that the slow flux through the chemical pathway is mainly limited by an unfavorable equilibrium at the Meisenheimer intermediate (EMc) formation and by a slow rate of hydrolysis of Arylated enzyme intermediate (EAr).;The EMc was examined by the Raman spectral analysis of the dehalogenase complexes formed with 4-FBA-CoA and 4-NBA-CoA. The relationship between active site polarizing forces through the H-bonding interaction of the backbone amide NH of Phe64, Gly114 acting on the benzoyl carbonyl group and the rate of formation of the EMc in the dehalogenase active site was determined. A linear correlation between C=O stretching frequency and "catalytic efficiency" was obtained.;The contribution of the active site Histidine 90 to catalysis of EAr hydrolysis was determined by kinetic analysis of His90 site directed mutants. A concerted mechanism for EAr hydrolysis was proposed due to the stabilization of phenolate anion leaving group by the polarization effect of the active site, resulting in shifting pKa of 4-HBA-CoA from 8.6 in water to less than 7 in the dehalogenase active site. This hypothesis was tested by studying the pKa of 4-HBA-CoA in D145A active site and Asp145-O-methybenzoyl-CoA ester adduct, formed by affinity labeling with 4-chloromethylbenzoyl CoA (4-McCIBA-CoA) in dehalogenase. The X-ray crystal structure of this adduct showed that the active site catalytic machinery was intact. The Raman spectral analysis showed that the ester C=O was polarized by the active site. Thus, the EAr hydrolysis occurs via a concerted mechanism where the contribution of base catalysis is modest.;160-fold, 1000-fold increased defluorination activity for 4-FBA-CoA was achieved by building a H-bond donor residue (AMS, or A86D) in the active site. The x-ray structural analysis of the A86S/T146A mutant enzyme reveals H-bond interaction between the hydroxyl group of Ser86 and the C (4) OH of 4-HBA-CoA substituent. 50100-fold increased denitrofication activity for 4-NBA-CoA was obtained by enlarging the dehalogenase active site.;Arthrobacter dehalogenase shares 47% identity with Pseudomonas dehalogenase. Steady state and transient state kinetic analysis have been conducted to compare the catalysis and specificity of these two enzymes.