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Identification of the histidine and aspartic acid residues essential for enzymatic activity of a family I.3 lipase by site‐directed mutagenesis
Author(s) -
Hyun-Ju Kwon,
Amada Kei,
Haruki Mitsuru,
Morikawa Masaaki,
Kanaya Shigenori
Publication year - 2000
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(00)02103-7
Subject(s) - histidine , lipase , catalytic triad , site directed mutagenesis , mutagenesis , biochemistry , enzyme , aspartic acid , circular dichroism , directed mutagenesis , chemistry , amino acid , mutation , stereochemistry , active site , biology , mutant , gene
A lipase from Pseudomonas sp. MIS38 (PML) is a member of the lipase family I.3. We analyzed the roles of the five histidine residues (His 30 , His 274 , His 291 , His 313 , and His 365 ) and five acidic amino acid residues (Glu 253 , Asp 255 , Asp 262 , Asp 275 , and Asp 290 ), which are fully conserved in the amino acid sequences of family I.3 lipases, by site‐directed mutagenesis. We showed that the mutation of His 313 or Asp 255 to Ala almost fully inactivated the enzyme, whereas the mutations of other residues to Ala did not seriously affect the enzymatic activity. Measurement of the far‐ and near‐UV circular dichroism spectra suggests that inactivation by the mutation of His 313 or Asp 255 is not due to marked changes in the tertiary structure. We propose that His 313 and Asp 255 , together with Ser 207 , form a catalytic triad in PML.