COMPREHENSIVE SITE-DIRECTED MUTAGENESIS OF L-2-HALO ACID DEHALOGENASETO PROBE CATALYTIC AMINO-ACID-RESIDUES

L-2-Halo acid dehalogenase catalyzes the stereospecific hydrolytic deh alogenation of L-2-halo acids, with inversion of the C-2-configuration . Seven L-2-halo acid dehalogenases from various bacterial strains are significantly similar to one another in their amino acid sequences (3 6-70% identity), and they are supposed to catalyze the reaction throug h the same mechanism. To identify catalytically important residues, we mutated all the 36 highly conserved charged and polar amino acid resi dues of L-2-halo acid dehalogenase from Pseudomonas sp. YL, which cons ists of 232 amino acid residues, by replacement of D by N, E by Q, R b y K, and vice versa, S and T by A, Y and W by F, M by L, and H by N. W e found that the replacement of D10, K151, S175, D180, R41, S118, T14, Y157, and N177 led to a significant loss in the enzyme activity or an increase in the K-m value for the substrate, showing their involvemen t in the catalysis. The roles of these residues are discussed.