Liang Zhao-Xun, Tsigos Iason, Lee Thomas, Bouriotis Vassilis, Resing Katheryn A, Ahn Natalie G, Klinman Judith P
Department of Chemistry, University of California, Berkeley, California 94720, USA.
Biochemistry. 2004 Nov 23;43(46):14676-83. doi: 10.1021/bi049004x.
The psychrophilic alcohol dehydrogenase (psADH) cloned from Antarctic Moraxella sp. TAE123 exhibits distinctive catalytic parameters in relation to the homologous thermophilic alcohol dehydrogenase (htADH) from Bacillus stearothermophilus LLD-R. Amide hydrogen-deuterium (H/D) exchange studies using Fourier-transformed infrared (FTIR) spectroscopy and mass spectrometry (MS) were conducted to investigate whether the differences are caused by variation in either global or regional protein flexibility. The FTIR H/D exchange study suggested that psADH does not share similar global flexibility with htADH at their physiologically relevant temperatures as has been predicted by the "corresponding state" hypothesis. However, the MS H/D exchange study revealed a more complicated picture concerning the flexibility of the two homologous enzymes. Analysis of the deuteration and exchange rates of protein-derived peptides suggested that only some functionally important regions in psADH that are involved in substrate and cofactor binding exhibit greater flexibility compared to htADH at low temperature (10 degrees C). These observations strongly suggest that variable conformational flexibility between the two protein forms is a local phenomenon, and that global H/D exchange measurement by FTIR can be misleading and should be used with discretion. These results are supportive of the idea that functionally important local flexibility can be uncoupled from global thermal stability. The structural factors underlying the differences in local protein flexibility and catalysis between htADH and psADH are discussed in conjunction with results from crystallographic and fluorescence spectroscopy studies.
从南极莫拉克斯氏菌属TAE123克隆的嗜冷醇脱氢酶（psADH）与嗜热脂肪芽孢杆菌LLD-R的同源嗜热醇脱氢酶（htADH）相比，具有独特的催化参数。利用傅里叶变换红外光谱（FTIR）和质谱（MS）进行了酰胺氢-氘（H/D）交换研究，以探究这些差异是否由整体或局部蛋白质柔韧性的变化引起。FTIR H/D交换研究表明，在其生理相关温度下，psADH与htADH不具有如“对应状态”假说所预测的相似整体柔韧性。然而，MS H/D交换研究揭示了这两种同源酶柔韧性方面更为复杂的情况。对蛋白质衍生肽段的氘化和交换速率分析表明，在低温（10摄氏度）下，psADH中仅一些参与底物和辅因子结合的功能重要区域比htADH表现出更大的柔韧性。这些观察结果有力地表明，两种蛋白质形式之间可变的构象柔韧性是一种局部现象，并且FTIR进行的整体H/D交换测量可能会产生误导，应谨慎使用。这些结果支持了功能重要的局部柔韧性可与整体热稳定性解耦的观点。结合晶体学和荧光光谱研究结果，讨论了htADH和psADH之间局部蛋白质柔韧性和催化差异背后的结构因素。
