REPOSITIONING THE CATALYTIC TRIAD ASPARTIC-ACID OF HALOALKANE DEHALOGENASE - EFFECTS ON STABILITY, KINETICS, AND STRUCTURE

Haloalkane dehalogenase (DhlA) catalyzes the hydrolysis of haloalkanes via an alkyl-enzyme intermediate. The covalent intermediate, which is formed by nucleophilic substitution with Asp124, is hydrolyzed by a w ater molecule that is activated by His289. The role of Asp260, which i s the third member of the catalytic triad, was studied by site-directe d mutagenesis. Mutation of Asp260 to asparagine resulted in a catalyti cally inactive D260N mutant, which demonstrates that the triad acid As p260 is essential for dehalogenase activity. Furthermore, Asp260 has a n important structural role, since the D260N enzyme accumulated mainly in inclusion bodies during expression, and neither substrate nor prod uct could bind in the active-site cavity. Activity for brominated subs trates was restored to D260N by replacing Asn148 with an aspartic or g lutamic acid. Both double mutants D260N+N148D and D260N+N148E had a 10 -fold reduced k(cat) and 40-fold higher K-m values for 1,2-dibromoetha ne compared to the wild-type enzyme. Pre-steady-state kinetic analysis of the D260N+N148E double mutant showed that the decrease in k(cat) w as mainly caused by a 220-fold reduction of the rate of carbon-bromine bond cleavage and a 10-fold decrease in the rate of hydrolysis of the alkyl-enzyme intermediate. On the other hand, bromide was released 12 -fold faster and via a different pathway than in the wild-type enzyme. Molecular modeling of the mutant showed that Glu148 indeed could take over the interaction with His289 and that there was a change in charg e distribution in the tunnel region that connects the active site with the solvent. On the basis of primary structure similarity between Dhl A and other alpha/beta-hydrolase fold dehalogenases, we propose that a conserved acidic residue at the equivalent position of Asn148 in DhlA is the third catalytic triad residue in the latter enzymes.