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 kcatfor hydride transfer, leading to a linear correlation between kcatand the weighted average exchange rate constant kHX( 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 Eactat high temperature. The data provide support for the hypothesis that protein dynamics play a key role in controlling hydrogen tunneling at enzyme active sites.