Dissecting the paradoxical effects of hydrogen bond mutations in the ketosteroid isomerase oxyanion hole

摘要: effects of these mutations and clarify the energetic importance Tyr16 hydrogen bond, we have determined 1.6-A resolution x-ray structure intermediate analogue, equilenin, bound to Tyr16Ser mutant measured rate mutating Ser, Thr, Ala, Gly. The nearly identical 200-fold reductions mutations, together with 6.4-A distance observed between Ser16 hydroxyl equilenin oxygens in structure, strongly suggest that more moderate effect this is not due maintenance a bond from Ser at position 16. These results, additional spectroscopic observations, prior structural studies Tyr16Phe mutation results unfavorable interactions dienolate beyond loss thereby exaggerating apparent benefit relative solution reaction. underscore complex energetics bonding sitedirected mutagenesis experiments.