期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2004
卷号：101
期号：26
页码：9556-9561
DOI：10.1073/pnas.0403337101
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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 {approx}30 and 45{degrees}C to attain a more dynamic conformational substate. Four of the five peptides exhibiting the transition above 40{degrees}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{degrees}C. By contrast, the five peptides exhibiting the transition at 30{degrees}C reside in the substrate-binding domain. This transition coincides with a change in the activation energy of kcat for hydride transfer, leading to a linear correlation between kcat and 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 Eact 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.
