Identification of active site residues essential to 4-chlorobenzoyl-coenzyme dehalogenase catalysis by chemical modification and site directed mutagenesis. Export

@article{Yang96, abstract = {4-Chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydrolysis of 4-CBA-CoA to 4-hydroxybenzoyl-coenzyme A (4-HBA-CoA) via a nucleophilic aromatic substitution pathway involving the participation of an active site carboxylate side chain in covalent catalysis. In this paper we report on the identification of conserved aspartate, histidine, and tryptophan residues essential to 4-CBA-CoA catalysis using chemical modification and site-directed mutagenesis techniques. Treatment of the dehalogenase with diethyl pyrocarbonate resulted in complete loss of catalytic activity (Kinact = 0.17 mM-1 min-1 at pH 6.5, 25 degrees C) that was fully regained by subsequent treatment with hydroxylamine. The protection from inactivation afforded by enzyme bound 4-HBA-CoA indicated that the essential histidine residues are located at the active site. Replacement of conserved histidine residues 81, 90, 94, and 208 with glutamine residues resul...}, address = {Department of Chemistry and Biochemistry, University of Maryland, College Park 20742, USA.}, author = {Yang, G. and Liu, R. Q. and Taylor, K. L. and Xiang, H. and Price, J. and Mariano, Dunaway D.}, citeulike-article-id = {388926}, editor = {1996/08/20}, journal = {Biochemistry}, keywords = {cat\_res, chemical\_modification, crystal\_structure, enzyme, mechanism, site\_directed\_mutagenesis}, number = {33}, posted-at = {2005-11-11 16:23:16}, priority = {2}, title = {Identification of active site residues essential to 4-chlorobenzoyl-coenzyme dehalogenase catalysis by chemical modification and site directed mutagenesis.}, volume = {35}, year = {1996} }

TY - JOUR ID - Yang96 L3 - citeulike-article-id:388926 N2 - 4-Chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydrolysis of 4-CBA-CoA to 4-hydroxybenzoyl-coenzyme A (4-HBA-CoA) via a nucleophilic aromatic substitution pathway involving the participation of an active site carboxylate side chain in covalent catalysis. In this paper we report on the identification of conserved aspartate, histidine, and tryptophan residues essential to 4-CBA-CoA catalysis using chemical modification and site-directed mutagenesis techniques. Treatment of the dehalogenase with diethyl pyrocarbonate resulted in complete loss of catalytic activity (Kinact = 0.17 mM-1 min-1 at pH 6.5, 25 degrees C) that was fully regained by subsequent treatment with hydroxylamine. The protection from inactivation afforded by enzyme bound 4-HBA-CoA indicated that the essential histidine residues are located at the active site. Replacement of conserved histidine residues 81, 90, 94, and 208 with glutamine residues resul... IS - 33 JF - Biochemistry AD - Department of Chemistry and Biochemistry, University of Maryland, College Park 20742, USA. TI - Identification of active site residues essential to 4-chlorobenzoyl-coenzyme dehalogenase catalysis by chemical modification and site directed mutagenesis. VL - 35 KW - cat_res KW - chemical_modification KW - crystal_structure KW - enzyme KW - mechanism KW - site_directed_mutagenesis AU - Yang, G AU - Liu, RQ AU - Taylor, KL AU - Xiang, H AU - Price, J AU - Mariano, Dunaway A2 - 1996/08/20 PY - 1996/// ER -