Investigation of the catalytic mechanism of the hotdog-fold enzyme superfamily Pseudomonas sp. strain CBS3 4-hydroxybenzoyl-CoA thioesterase.
The 4-hydroxybenzoyl-CoA (4-HB-CoA) thioesterase from Pseudomonas sp. strain CBS3 catalyzes the final step of the 4-chlorobenzoate degradation pathway, which is the hydrolysis of 4-HB-CoA to coenzyme A (CoA) and 4-hydroxybenzoate (4-HB). In previous work, X-ray structural analysis of the substrate-bound thioesterase provided evidence of the role of an active site Asp17 in nucleophilic catalysis [Thoden, J. B., Holden, H. M., Zhuang, Z., and Dunaway-Mariano, D. (2002) X-ray crystallographic analyses of inhibitor and substrate complexes of wild-type and mutant 4-hydroxybenzoyl-CoA thioesterase. J. Biol. Chem. 277, 27468-27476]. In the study presented here, kinetic techniques were used to test the catalytic mechanism that was suggested by the X-ray structural data. The time course for the multiple-turnover reaction of 50 μM [(14)C]-4-HB-CoA catalyzed by 10 μM thioesterase supported a two-step pathway in which the second step is rate-limiting. Steady-state product inhibition studies revealed that binding of CoA (K(is) = 250 ± 70 μM; K(ii) = 900 ± 300 μM) and 4-HB (K(is) = 1.2 ± 0.2 mM) is weak, suggesting that product release is not rate-limiting. A substantial D(2)O solvent kinetic isotope effect (3.8) on the steady-state k(cat) value (18 s(-1)) provided evidence that a chemical step involving proton transfer is the rate-limiting step. Taken together, the kinetic results support a two-chemical pathway. The microscopic rate constants governing the formation and consumption of the putative aspartyl 17-(4-hydroxybenzoyl)anhydride intermediate were determined by simulation-based fitting of a kinetic model to time courses for the substrate binding reaction (5.0 μM 4-HB-CoA and 0.54 μM thioesterase), single-turnover reaction (5 μM [(14)C]-4-HB-CoA catalyzed by 50 μM thioesterase), steady-state reaction (5.2 μM 4-HB-CoA catalyzed by 0.003 μM thioesterase), and transient-state multiple-turnover reaction (50 μM [(14)C]-4-HB-CoA catalyzed by 10 μM thioesterase). Together with the results obtained from solvent (18)O labeling experiments, the findings are interpreted as evidence of the formation of an aspartyl 17-(4-hydroxybenzoyl)anhydride intermediate that undergoes rate-limiting hydrolytic cleavage at the hydroxybenzoyl carbonyl carbon atom.
Department of Chemistry and Chemical Biology, University of New Mexico, Albuquerque, New Mexico 87131, United States.
Zhihao
Z
Zhuang
John
J
Latham
Feng
F
Song
Wenhai
W
Zhang
Michael
M
Trujillo
Debra
D
Dunaway-Mariano
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D002723:Chlorobenzoates / Q000737:chemistry / Q000378:metabolism; D003065:Coenzyme A / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D006868:Hydrolysis; D008024:Ligands; D010226:Parabens / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D055503:Protein Multimerization; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D002723:Chlorobenzoates; D008024:Ligands; D010226:Parabens; C092167:4-hydroxybenzoyl-CoA hydrolase; D013869:Thiolester Hydrolases; C040768:4-chlorobenzoic acid; C038193:4-hydroxybenzoic acid; D003065:Coenzyme A