title: Kinetic, Raman, NMR, and site-directed mutagenesis studies of the Pseudomonas sp. strain CBS3 4-hydroxybenzoyl-CoA thioesterase active site.
abstract: 4-Hydroxybenzoyl-coenzyme A (4-HBA-CoA) thioesterase catalyzes the hydrolysis of 4-HBA-CoA to 4-hydroxybenzoate and CoA. X-ray crystallographic analysis of the liganded enzyme has shown that the benzoyl thioester and pantetheine moieties of the substrate ligand are bound in a narrow crevice while the nucleotide moiety rests on the protein surface (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., in press). Asp17 is positioned in the crevice, close to the substrate thioester C=O, which in turn interacts with the positive pole of an alpha-helix macrodipole. In this paper we report the results from spectral, mutagenesis, and kinetic studies which show (1) that substrate activation involves restricted thioester C=O conformational freedom and a modest enhancement of C=O bond polarization, (2) that the nucleotide unit of the substrate is bound through interaction with the protein surface, and (3) that Asp17 contributes a rate factor of 10(4), consistent with its proposed role of general base or nucleophile.
authors.affiliation: Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
authors.forename: Zhihao
authors.initials: Z
authors.lastname: Zhuang
authors.forename: Feng
authors.initials: F
authors.lastname: Song
authors.forename: Wenhai
authors.initials: W
authors.lastname: Zhang
authors.forename: Kimberly
authors.initials: K
authors.lastname: Taylor
authors.forename: Angela
authors.initials: A
authors.lastname: Archambault
authors.forename: Debra
authors.initials: D
authors.lastname: Dunaway-Mariano
authors.forename: Jian
authors.initials: J
authors.lastname: Dong
authors.forename: Paul R
authors.initials: PR
authors.lastname: Carey
mesh_terms: D000225:Adenine / Q000737:chemistry; D019943:Amino Acid Substitution / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics; D002384:Catalysis; D003065:Coenzyme A / Q000737:chemistry; D006860:Hydrogen Bonding; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010204:Pantetheine / Q000737:chemistry; D010759:Phosphorus Isotopes; D011485:Protein Binding / Q000235:genetics; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D013059:Spectrum Analysis, Raman; D055672:Static Electricity; D013379:Substrate Specificity / Q000235:genetics; D013869:Thiolester Hydrolases / Q000737:chemistry* / Q000235:genetics*
chemical_list: D010759:Phosphorus Isotopes; D001224:Aspartic Acid; D010204:Pantetheine; C092167:4-hydroxybenzoyl-CoA hydrolase; D013869:Thiolester Hydrolases; D000225:Adenine; D003065:Coenzyme A