Effects of the residue adjacent to the reactive serine on the substrate interactions ofDrosophila esterase 6 |
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Authors: | Mark A Myers Marion J Healy John G Oakeshott |
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Institution: | (1) CSIRO Division of Entomology, GPO Box 1700, ACT 2601 Canberra, Australia;(2) Botany Department, Australian National University, ACT 2601 Canberra, Australia;(3) Monash University Department of Medicine, Box Hill Hospital, Nelson Road, 3128 Box Hill, Victoria, Australia |
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Abstract: | Esterase 6 fromDrosophila melanogaster is a carboxylesterase that belongs to the serine esterase multigene family. It has a basic histidine (His) at residue 187,
adjacent to the reactive serine (Ser) at residue 188, whereas most other characterized members of the family have an acidic
glutamate (Glu) in the equivalent position. We have used site-directedin vitro mutagenesis to replace the His codon of the esterase 6 gene with either Gln or Glu codons. The enzymes encoded by these active-site
mutants and a wild-type control have been expressed, purified, and characterized. Substitution of Gln for His at position
187 has little effect on the biochemical properties of esterase 6, but the presence of Glu at this position is associated
with three major differences. First, the pH optimum is increased from 7 to 9. Second, the mutant enzyme shows decreased activity
for β-naphthyl esters andp-nitrophenyl acetate but has gained the ability to hydrolyze acetylthiocholine. Finally, the Gibb’s free energy of activation
for the enzyme is increased. These results suggest that residue 187 interacts directly with the substrate alkyl group and
that this interaction is fully realized in the transition state. We further propose that the presence of His rather than Glu
at position 187 in esterase 6 contributes significantly to its functional divergence from the cholinesterases and that this
divergence is due to different interactions between residue 187 and the substrate alkyl group. |
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Keywords: | serine esterase substrate interactions Drosophila acetylcholine |
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