| Autor: |
Ciulla DA; Chemistry Department, Binghamton University (SUNY) , Binghamton, New York 13902, United States., Jorgensen MT; Department of Chemistry, SUNY-ESF , Syracuse, New York 13210, United States., Giner JL; Department of Chemistry, SUNY-ESF , Syracuse, New York 13210, United States., Callahan BP; Chemistry Department, Binghamton University (SUNY) , Binghamton, New York 13902, United States. |
| Abstrakt: |
Proteins in the hedgehog family undergo self-catalyzed endoproteolysis involving nucleophilic attack by a molecule of cholesterol. Recently, a conserved aspartate residue (D303, or D46) of hedgehog was identified as the general base that activates cholesterol during this unusual autoprocessing event; mutation of the catalyzing functional group (D303A) reduces activity by >10 4 -fold. Here we report near total rescue of this ostensibly dead general base mutant by a synthetic substrate, 3β-hydroperoxycholestane (3HPC) in which the sterol -OH group is replaced by the hyper nucleophilic -OOH group. Other hedgehog point mutants at D303, also unreactive with cholesterol, accepted 3HPC as a substrate with the rank order: WT > D303A ≈ D303N ≫ D303R, D303E. We attribute the revived activity with 3-HPC to the α-effect, where tandem electronegative atoms exhibit exceptionally high nucleophilicity despite relatively low basicity. |
