bi9609533_si_001.pdf (162.78 kB)
Identification of Active Site Residues Essential to 4-Chlorobenzoyl−Coenzyme A Dehalogenase Catalysis by Chemical Modification and Site Directed Mutagenesis†
journal contribution
posted on 1996-08-20, 00:00 authored by Guang Yang, Rui-Qin Liu, Kimberly L. Taylor, Hong Xiang, Jack Price, Debra Dunaway-Mariano4-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 °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 resulted in a significant loss of
catalytic activity only in the cases of the
histidine 81 and 90 mutants. Substrate and product ligand binding
studies showed that binding is not
significantly inhibited in these mutants. Site directed
mutagenesis of a selection of conserved aspartate
and glutamate residues, identified aspartate 145 as being essential to
dehalogenase catalysis. Ligand
binding studies showed that this residue is not required for tight
substrate/product binding. Chemical
modification of the dehalogenase with N-bromosuccinimide
resulted in full loss of catalytic activity that
was prevented by saturation of the active site with product ligand,
providing evidence favoring an essential
active site tryptophan. Phenylalanine replacement of conserved
tryptophan residues 179 and 137 reduced
catalytic activity only in the latter (kcat =
0.03% of wild-type dehalogenase). On the basis of these
results
and the recently determined 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.,
Xiang, H., Wesenberg, G., Dunaway-Mariano, D., Holden, H. M. (1996) Biochemistry 35,
8103−8109] we propose that aspartate 145 functions
as the active site nucleophile, that tryptophan 137 serves as a
hydrogen bond donor to the aspartate 145
CO, and that histidine 90 serves to deprotonate the bound
H2O molecule.