Farnum M F, Magde D, Howell E E, Hirai J T, Warren M S, Grimsley J K, Kraut J
Department of Chemistry, University of California, San Diego, La Jolla 92093.
Biochemistry. 1991 Dec 10;30(49):11567-79. doi: 10.1021/bi00113a012.
A remarkable correlation has been discovered between fluorescence lifetimes of bound NADPH and rates of hydride transfer among mutants of dihydrofolate reductase (DHFR) from Escherichia coli. Rates of hydride transfer from NADPH to dihydrofolate change by a factor of 1,000 for the series of mutant enzymes. Since binding constants for the initial complex between coenzyme and DHFR change by only a factor of 10, the major portion of the change in hydride transfer must be attributed to losses in transition-state stabilization. The time course of fluorescence decay for NADPH bound to DHFR is biphasic. Lifetimes ranging from 0.3 to 0.5 ns are attributed to a solvent-exposed dihydronicotinamide conformation of bound coenzyme which is presumably not active in catalysis, while decay times (tau 2) in the range of 1.3 to 2.3 ns are assigned to a more tightly bound species of NADPH in which dihydronicotinamide is sequestered from solvent. It is this slower component that is of interest. Ternary complexes with three different inhibitors, methotrexate, 5-deazafolate, and trimethoprim, were investigated, along with the holoenzyme complex; 3-acetylNADPH was also investigated. Fluorescence polarization decay, excitation polarization spectra, the temperature variation of fluorescence lifetimes, fluorescence amplitudes, and wavelength of absorbance maxima were measured. We suggest that dynamic quenching or internal conversion promotes decay of the excited state in NADPH-DHFR. When rates of hydride transfer are plotted against the fluorescence lifetime (tau 2) of tightly bound NADPH, an unusual correlation is observed. The fluorescence lifetime becomes longer as the rate of catalysis decreases for most mutants studied. However, the fluorescence lifetime is unchanged for those mutations that principally alter the binding of dihydrofolate while leaving most dihydronicotinamide interactions relatively undisturbed. The data are interpreted in terms of possible dynamic motions of a flexible loop region in DHFR which closes over both substrate and coenzyme binding sites. These motions could lead to faster rates of fluorescence decay in holoenzyme complexes and, when correlated over time, may be involved in other motions which give rise to enhanced rates of catalysis in DHFR.
在来自大肠杆菌的二氢叶酸还原酶（DHFR）突变体中，已发现结合的NADPH的荧光寿命与氢化物转移速率之间存在显著相关性。对于一系列突变酶，从NADPH到二氢叶酸的氢化物转移速率变化了1000倍。由于辅酶与DHFR之间初始复合物的结合常数仅变化了10倍，氢化物转移变化的主要部分必须归因于过渡态稳定性的损失。与DHFR结合的NADPH的荧光衰减时间进程是双相的。0.3至0.5纳秒的寿命归因于结合辅酶的溶剂暴露的二氢烟酰胺构象，该构象可能在催化中无活性，而1.3至2.3纳秒范围内的衰减时间（tau 2）则归因于一种结合更紧密的NADPH物种，其中二氢烟酰胺与溶剂隔离。正是这种较慢的成分令人感兴趣。研究了与三种不同抑制剂甲氨蝶呤、5-脱氮叶酸和甲氧苄啶形成的三元复合物，以及全酶复合物；还研究了3-乙酰基NADPH。测量了荧光偏振衰减、激发偏振光谱、荧光寿命的温度变化、荧光振幅和吸收最大值波长。我们认为动态猝灭或内转换促进了NADPH-DHFR中激发态的衰减。当将氢化物转移速率与紧密结合的NADPH的荧光寿命（tau 2）作图时，观察到一种不寻常的相关性。对于大多数研究的突变体，随着催化速率降低，荧光寿命变长。然而，对于那些主要改变二氢叶酸结合而使大多数二氢烟酰胺相互作用相对不受干扰的突变，荧光寿命不变。数据根据DHFR中一个柔性环区域可能的动态运动来解释，该区域封闭底物和辅酶结合位点。这些运动可能导致全酶复合物中荧光衰减速率加快，并且当随时间相关时，可能参与其他运动，从而导致DHFR中催化速率提高。
