title: Comparative analysis of two different amide-to-ester bond mutations in the beta-sheet of 4-oxalocrotonate tautomerase.
abstract: Here we describe the total chemical synthesis and biophysical characterization of two backbone-modified, ester bond-containing analogues of the homohexameric enzyme 4-oxalocrotonate tautomerase (4OT). The amide-to-ester bond mutations in the two analogues in this study, (OI2)4OT and (OI7)4OT, were designed to effectively delete specific backbone-backbone hydrogen bonds in the beta-sheet region of the native 4OT hexamer. The (OI2)4OT and (OI7)4OT analogues each contained one ester bond per monomer that effectively deleted 12 backbone-backbone hydrogen bonds per hexamer. The structural properties of each analogue were characterized by size-exclusion chromatography (SEC), far-UV CD spectroscopy, and catalytic activity measurements, and they were found to be very similar to the structural properties of the wild-type enzyme. The results of equilibrium unfolding studies revealed that the (OI2)4OT and (OI7)4OT analogues were stabilized by 47.7 +/- 2.5 and 45.0 +/- 2.5 kcal/mol, respectively, under standard state conditions (1 M hexamer) as compared to a value of 69.6 +/- 3.3 kcal/mol for the wild-type control. Our results suggest that the two different, but structurally similar, backbone-backbone hydrogen bonds deleted in (OI2)4OT and (OI7)4OT make nearly equivalent contributions to the thermodynamic stability of the 4OT hexamer.
authors.affiliation: Department of Chemistry, Duke University, Durham, North Carolina 27708-0346, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Silinski
authors.forename: Michael C
authors.initials: MC
authors.lastname: Fitzgerald
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D019791:Guanidine / Q000494:pharmacology; D006860:Hydrogen Bonding; D007535:Isomerases / Q000737:chemistry* / Q000235:genetics*; D009154:Mutation; D010452:Peptide Biosynthesis; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013997:Time Factors; D014466:Ultraviolet Rays
chemical_list: D000596:Amino Acids; C021918:4-oxalocrotonate tautomerase; D007535:Isomerases; D019791:Guanidine