REACTION-MECHANISM OF L-2-HALOACID DEHALOGENASE OF PSEUDOMONAS SP. YL- IDENTIFICATION OF ASP(10) AS THE ACTIVE-SITE NUCLEOPHILE BY O-18 INCORPORATION EXPERIMENTS

L-2-Haloacid dehalogenase (EC 3.8.1.2) catalyzes the hydrolytic dehalo genation of L-2-haloacids to produce the corresponding D-2-hydroxy aci ds, We have analyzed the reaction mechanism of the enzyme from Pseudom onas sp, YL and found that Asp(10) is the active site nucleophile, Whe n the multiple turnover enzyme reaction was carried out in (H2O)-O-18 with L-2-chloropropionate as a substrate, lactate produced was labeled with O-18. However, when the single turnover enzyme reaction was carr ied out by use of a large excess of the enzyme, the product was not la beled, This suggests that an oxygen atom of the solvent water is first incorporated into the enzyme and then transferred to the product, Aft er the multiple turnover reaction in (H2O)-O-18, the enzyme was digest ed with lysyl endopeptidase, and the molecular masses of the peptide f ragments formed were measured by an ionspray mass spectrometer, Two O- 18 atoms were shown to be incorporated into a hexapeptide, Gly(6)-Lys( 11). Tandem mass spectrometric analysis of this peptide revealed that Asp(10) was labeled with two O-18 atoms, Our previous site-directed mu tagenesis experiment showed that the replacement of Asp(10) led to a s ignificant loss in the enzyme activity. These results indicate that As p(10) acts as a nucleophile on the alpha-carbon of the substrate leadi ng to the formation of an ester intermediate, which is hydrolyzed by n ucleophilic attack of a water molecule on the carbonyl carbon atom.