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Effect of site‐directed mutagenesis on conserved positions of Drosophila alcohol dehydrogenase
Author(s) -
Cols Neus,
Marfany Gemma,
Atrian Sílvia,
Gonzàlez-Duarte Roser
Publication year - 1993
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 - 10.1016/0014-5793(93)80043-t
Subject(s) - mutagenesis , mutant , alcohol dehydrogenase , drosophila (subgenus) , site directed mutagenesis , biochemistry , enzyme , mutation , conserved sequence , biology , drosophila melanogaster , genetics , chemistry , gene , peptide sequence
Tyr 152 and Lys 156 may be functionally important residues in Drosophila ADH as they are conserved in the genus and in all short‐chain dehydrogenases. In addition, unaltered Gly positions could have a crucial role in the building of the structural framework. We have modified Drosophila ADH and expressed the mutant forms in E. coli . Mutation of Tyr 152 to Glu or Gin, Lys 156 to Ile, Gly 184 to Leu, and the double mutant Gly 130 to Cys and Gly 133 to Ile, all rendered, with different substrates and at different pHs, an inactive enzyme. Results suggest that Tyr 152 and Lys 156 are involved in catalysis and that Gly 130 , Gly 133 and Gly 184 contribute substantially to the structure of the active form.