Department of Chemistry, University of Iowa, Iowa City, Iowa 52242, United States.
J Phys Chem B. 2012 Jan 12;116(1):542-8. doi: 10.1021/jp208677u. Epub 2011 Dec 15.
Functionally relevant femtosecond to picosecond dynamics in enzyme active sites can be difficult to measure because of a lack of spectroscopic probes that can be located in the active site without altering the behavior of the enzyme. We have developed a new NAD(+) analog 3-Picolyl Azide Adenine Dinucleotide (PAAD(+)), which has the potential to be a general spectroscopic probe for NAD-dependent enzymes. This analog is stable and binds in the active site of a typical NAD-dependent enzyme formate dehydrogenase (FDH) with characteristics similar to those of natural NAD(+). It has an isolated infrared transition with high molar absorptivity that makes it suitable for observing enzyme dynamics using 2D IR spectroscopy. 2D IR experiments show that in aqueous solution, the analog undergoes complete spectral diffusion within hundreds of femtoseconds consistent with the water hydrogen bonding dynamics that would be expected. When bound to FDH in a binary complex, it shows picosecond fluctuations and a large static offset, consistent with previous studies of the binary complexes of this enzyme. These results show that PAAD(+) is an excellent probe of local dynamics and that it should be a general tool for probing the dynamics of a wide range of NAD-dependent enzymes.
在酶活性中心，功能相关的飞秒到皮秒动力学很难测量，因为缺乏可以在不改变酶行为的情况下定位在活性中心的光谱探针。我们开发了一种新的 NAD(+)类似物 3-吡啶基叠氮基腺嘌呤二核苷酸（PAAD(+)），它有可能成为一种用于 NAD 依赖性酶的通用光谱探针。该类似物稳定，能够结合在典型的 NAD 依赖性酶甲酸脱氢酶（FDH）的活性中心，其特征与天然 NAD(+)相似。它具有孤立的红外跃迁，摩尔吸光率高，适合使用 2D IR 光谱观察酶动力学。2D IR 实验表明，在水溶液中，该类似物在数百飞秒内经历完全的光谱扩散，与预期的水氢键动力学一致。当与 FDH 在二元复合物中结合时，它表现出皮秒波动和大的静态偏移，与该酶的二元复合物的先前研究一致。这些结果表明，PAAD(+)是局部动力学的极好探针，它应该是探测广泛的 NAD 依赖性酶动力学的通用工具。
