Hydrogen-deuterium exchange of a primary amide as a model for asparagine and glutamine exchange in proteins

Hydrogen-deuterium exchange of the primary amide, isobutyramide, was investigated as a model for asparagine and glutamine-NH₂ exchange in a protein. A simple amide was chosen since the structures of several well-characterized proteins show most of these residues to be exposed to solvent. Isobutyramide-exchange data were obtained in 1:1 D₂O:dioxane solutions using a near-infrared method. The rate data were strictly pseudo-first order and yielded an average of 95% exchange of the primary amide hydrogens. In analogy with secondary amides, the pD dependence of the rate constants was characteristic of specific acid and base catalysis. In addition, analysis of the rate-pD profile for isobutyramide indicated a significant uncatalyzed exchange reaction. Temperature-dependence studies of the first-order rate constants at a fixed pD yielded an apparent activation energy of 19.3 kcal/mole. Predicted half-life times for the exposed primary amide hydrogens in proteins, based on these exchange parameters, indicate that asparagine and glutamine side chains generally would contribute to the overall rate data only below 15°C and then only for approximately 1 pD unit around the point of minimum reaction velocity.