IDENTIFICATION OF ACTIVE-SITE RESIDUES ESSENTIAL TO 4-CHLOROBENZOYL-COENZYME-A DEHALOGENASE CATALYSIS BY CHEMICAL MODIFICATION AND SITE-DIRECTED MUTAGENESIS

4-Chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydr olysis of 4-CBA-CoA to 4-hydroxybenzoyl-coenzyme A (4-HBA-CoA) via a n ucleophilic aromatic substitution pathway involving the participation of an active site carboxylate side chain in covalent catalysis. In thi s paper we report on the identification of conserved aspartate, histid ine, and tryptophan residues essential to 4-CBA-CoA catalysis using ch emical modification and site-directed mutagenesis techniques. Treatmen t of the dehalogenase with diethyl pyrocarbonate resulted in complete loss of catalytic activity (k(inact) = 0.17 mM(-1) min(-1) at pH 6.5, 25 degrees C) that was fully regained by subsequent treatment with hyd roxylamine. 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 resulted in a significant loss o f catalytic activity only in the cases of the histidine 81 and 90 muta nts. Substrate and product ligand binding studies showed that binding is not significantly inhibited in these mutants. Site directed mutagen esis of a selection of conserved aspartate and glutamate residues, ide ntified aspartate 145 as being essential to dehalogenase catalysis. Li gand binding studies showed that this residue is not required for tigh t substrate/product binding. Chemical modification of the dehalogenase with N-bromosuccinimide resulted in full loss of catalytic activity t hat was prevented by saturation of the active site with product ligand , providing evidence favoring an essential active site tryptophan. Phe nylalanine replacement of conserved tryptophan residues 179 and 137 re duced catalytic activity only in the latter (k(cat) = 0.03% of wild-ty pe dehalogenase). On the basis of these results and the recently deter mined X-ray crystal structure of the complex of 4-CBA-CoA dehalogenase and 4-HBA-CoA [Benning, M. M., Taylor, K. L., Liu, R.-Q., Yang, G., X iang, H., Wesenberg, G., Dunaway-Mariano, D., Holden, H. M. (1996) Bio chemistry 35, 8103-8109] we propose that aspartate 145 functions as th e active site nucleophile, that tryptophan 137 serves as a hydrogen bo nd donor to the aspartate 145 C=O, and that histidine 90 serves to dep rotonate the bound H2O molecule.