3 years ago

Chemical Bypass of General Base Catalysis in Hedgehog Protein Cholesterolysis Using a Hyper-Nucleophilic Substrate

Chemical Bypass of General Base Catalysis in Hedgehog Protein Cholesterolysis Using a Hyper-Nucleophilic Substrate
Brian P. Callahan, Michael T. Jorgensen, Daniel A. Ciulla, José-Luis Giner
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 >104-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.

Publisher URL: http://dx.doi.org/10.1021/jacs.7b05161

DOI: 10.1021/jacs.7b05161

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