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 approximate to30 and 45degreesC to attain a more dynamic conformational substate. Four of the five peptides exhibiting the transition above WC 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 >45degreesC. By contrast, the five peptides exhibiting the transition at 30degreesC 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.