| Autor: |
Zhao J, Ciulla DA; Department of Chemistry , Binghamton University, State University of New York , Binghamton , New York 13902 , United States., Xie J, Wagner AG; Department of Chemistry , Binghamton University, State University of New York , Binghamton , New York 13902 , United States., Castillo DA; Department of Chemistry , Binghamton University, State University of New York , Binghamton , New York 13902 , United States., Zwarycz AS, Lin Z, Beadle S; Department of Chemistry , State University of New York-ESF , Syracuse , New York 13210 , United States., Giner JL; Department of Chemistry , State University of New York-ESF , Syracuse , New York 13210 , United States., Li Z; Wadsworth Center , New York State Department of Health , Albany , New York 12201 , United States., Li H; Wadsworth Center , New York State Department of Health , Albany , New York 12201 , United States.; Department of Biomedical Sciences, School of Public Health , University at Albany , Albany , New York 12222 , United States., Banavali N; Wadsworth Center , New York State Department of Health , Albany , New York 12201 , United States.; Department of Biomedical Sciences, School of Public Health , University at Albany , Albany , New York 12222 , United States., Callahan BP; Department of Chemistry , Binghamton University, State University of New York , Binghamton , New York 13902 , United States., Wang C |
| Abstrakt: |
Hedgehog (Hh) autoprocessing converts Hh precursor protein to cholesterylated Hh ligand for downstream signaling. A conserved active-site aspartate residue, D46, plays a key catalytic role in Hh autoprocessing by serving as a general base to activate substrate cholesterol. Here we report that a charge-altering Asp-to-His mutant (D46H) expands native cholesterylation activity and retains active-site conformation. Native activity toward cholesterol was established for D46H in vitro using a continuous FRET-based autoprocessing assay and in cellulo with stable expression in human 293T cells. The catalytic efficiency of cholesterylation with D46H is similar to that with wild type (WT), with k max / K M = 2.1 × 10 3 and 3.7 × 10 3 M -1 s -1 , respectively, and an identical p K a = 5.8 is obtained for both residues by NMR. To our knowledge this is the first example where a general base substitution of an Asp for His preserves both the structure and activity as a general base. Surprisingly, D46H exhibits increased catalytic efficiency toward non-native substrates, especially coprostanol (>200-fold) and epicoprostanol (>300-fold). Expanded substrate tolerance is likely due to stabilization by H46 of the negatively charged tetrahedral intermediate using electrostatic interactions, which are less constrained by geometry than H-bond stabilization by D46. In addition to providing fundamental insights into Hh autoprocessing, our findings have important implications for protein engineering and enzyme design. |
