Thermal-activated protein mobility and its correlation with catalysis in thermophilic alcohol dehydrogenase

Temperature-dependent hydrogen-deuterium (H/D) exchange of the thermophilic alcohol dehydrogenase (htADH) has been studied by using liquid chromatography-coupled mass spectrometry. Analysis of the changes in H/D exchange patterns for the protein-derived peptides suggests that some regions of htADH are in a rigid conformational substate at reduced temperatures with limited cooperative protein motion. The enzyme undergoes two discrete transitions at ≈30 and 45°C to attain a more dynamic conformational substate. Four of the five peptides exhibiting the transition above 40°C are in direct contact with the cofactor, and the NAD⁺-binding affinity is also altered in this temperature range, implicating a change in the mobility of the cofactor-binding domain >45°C. By contrast, the five peptides exhibiting the transition at 30°C reside in the substrate-binding domain. This transition coincides with a change in the activation energy of k _cat for hydride transfer, leading to a linear correlation between k_cat and the weighted average exchange rate constant k _HX(WA) for the five peptides. These observations indicate a direct coupling between hydride transfer and protein mobility in htADH, and that an increased mobility is at least partially responsible for the reduced E _act at high temperature. The data provide support for the hypothesis that protein dynamics play a key role in controlling hydrogen tunneling at enzyme active sites.