CRYSTAL-STRUCTURES OF REACTION INTERMEDIATES OF L-2-HALOACID DEHALOGENASE AND IMPLICATIONS FOR THE REACTION-MECHANISM

Crystal structures of L-2-haloacid dehalogenase from Pseudomonas sp, Y L complexed with monochloroacetate, L-2-chlorobutyrate, L-2-chloro-3-m ethylbutyrate, or L-2-chloro-4-methylvalerate were determined at 1.83- , 2.0-, 2.2-, and 2.2-Angstrom resolutions, respectively, using the co mplex crystals prepared with the S175A mutant, which are isomorphous w ith those of the wild-type enzyme, These structures exhibit unique str uctural features that correspond to those of the reaction intermediate s. In each case, the nucleophile Asp-10 is esterified with the dechlor inated moiety of the substrate. The substrate moieties in all but the monochloroacetate intermediate have a D-configuration at the C-2 atom. The overall polypeptide fold of each of the intermediates is similar to that of the wild-type enzyme. However, it is clear that the Asp-10- Ser-20 region moves to the active site in all of the intermediates, an d the Tyr-91-Asp-102 and Leu-117-Arg-135 regions make conformational c hanges in all but the monochloroacetate intermediates. Ser-118 is loca ted near the carboxyl group of the substrate moiety; this residue prob ably serves as a binding residue for the substrate carboxyl group. The hydrophobic pocket, which is primarily composed of the Tyr-12, Gln-42 , Leu-45, Phe-60, Lys-151, Asn-177, and Trp-179 side chains, exists ar ound the alkyl group of the substrate moiety, This pocket may play an important role in stabilizing the alkyl group of the substrate moiety through hydrophobic interactions, and may also play a role in determin ing the stereospecificity of the enzyme. Moreover, a water molecule, w hich is absent in the substrate-free enzyme, is present in the vicinit ies of the carboxyl carbon of Asp-10 and the side chains of Asp-180, A sn-177, and Ala-175 in each intermediate. This water molecule may hydr olyze the ester intermediate and its substrate. These findings crystal lographically demonstrate that the enzyme reaction proceeds through th e formation of an ester intermediate with the enzyme's nucleophile Asp -10.