NMR evidence for the participation of a low-barrier hydrogen bond in the mechanism of delta 5-3-ketosteroid isomerase

摘要: Abstract Delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) promotes an allylic rearrangement involving intramolecular proton transfer via a dienolic intermediate. This enzyme enhances the catalytic rate by factor of 10(10). Two residues, Tyr-14, general acid that polarizes steroid 3-carbonyl group and facilitates enolization, Asp-38 base abstracts transfers 4 beta-proton to 6 beta-position, contribute 10(4.7) 10(5.6) increase, respectively. A major mechanistic enigma is huge disparity between pKa values groups their targets. Upon binding analog dienolate intermediate isomerase, NMR detects highly deshielded resonance at 18.15 ppm in proximity aromatic protons, with 3-fold preference for protium over deuterium (fractionation factor, phi = 0.34), consistent formation short, strong (low-barrier) hydrogen bond Tyr-14. The strength this estimated be least 7.1 kcal/mol. relatively inaccessible bulk solvent pH insensitive. Low-barrier bonding Tyr-14 intermediate, conjunction previously demonstrated tunneling contribution Asp-38, provide plausible quantitative explanation high power isomerase.