Structural equilibrium fluctuations in mesophilic and thermophilic alpha-amylase

By comparing a mesophilic alpha-amylase with its thermophilic homolog, we i nvestigated the relationship between thermal stability and internal equilib rium fluctuations. Fourier transform infrared spectroscopy monitoring hydro gen/deuterium (H/D) exchange kinetics and incoherent neutron scattering mea suring picosecond dynamics were used to study dynamic features of the folde d state at room temperature. Fairly similar rates of slowly exchanging amid e protons indicate about the same free energy of stabilization Delta G(stab ) for both enzymes at room temperature. With respect to motions on shorter time scales, the thermophilic enzyme is characterized by an unexpected high er structural flexibility as compared to the mesophilic counterpart. In par ticular, the picosecond dynamics revealed a higher degree of conformational freedom for the thermophilic alpha-amylase. The mechanism proposed for inc reasing thermal stability in the present case is characterized by entropic stabilization and by flattening of the curvature of Delta G(stab) as a func tion of temperature.