Low energy of activation for amide hydrogen exchange reactions in proteins supports a local unfolding model.

Hydrogen exchange reactions of amides in hen egg white lysozyme that are pH dependent and have a low energy of activation have been shown to be in accordance with a reaction mechanism in two steps, an equilibrium step and an exchange step. These results are not in agreement with the model, proposed by C.K. Woodward & B.D. Hilton, known as the penetration model. Therefore our results suggest that this model should be revised. The amide hydrogen/deuterium exchange rates in hen egg white lysozyme were measured at 4 degrees C, 10 degrees C, 15 degrees C and 25 degrees C at pH 7.0 by 1H nuclear magnetic resonance spectroscopy. Activation energies of the exchange reactions in the range from 20 kJ mol-1 to 333 kJ mol-1 were obtained for 32 of the 129 residues in the protein. The amides of lysozyme studied here could be divided into two groups, one group of amides are characterized by an observed amide exchange rate (ko) in the range 10(-4) to 10(-6) s-1, an equilibrium constant k1/k2 close to 10(-5), a low energy of activation (20 to 50 kJ mol-1) and a distance less than 6 A from solvent. The other group of amides are characterized by a ko less than 10(-6) s-1, a k1/k2 close to 10(-7), higher energies of activation (40 to 330 kJ mol-1) and a distance more than 4 A from solvent. In terms of structure the amides of the last group are from the core of the protein. They are typically involved in a hydrogen bond and form part of the secondary structure either as interior alpha-helices or central strands of beta-sheets. The first group consists of amides that are in the shell of the protein between the core and the surface. These amides are typically hydrogen bonded and involved in secondary structure such as external alpha-helices or outer strands of beta-sheets and turns.