Dong J, Drohat A C, Stivers J T, Pankiewicz K W, Carey P R
Department of Biochemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106-4935, USA.
Biochemistry. 2000 Oct 31;39(43):13241-50. doi: 10.1021/bi001437m.
Using off-resonance Raman spectroscopy, we have examined each complex along the catalytic pathway of the DNA repair enzyme uracil DNA glycosylase (UDG). The binding of undamaged DNA to UDG results in decreased intensity of the DNA Raman bands, which can be attributed to an increased level of base stacking, with little perturbation in the vibrational modes of the DNA backbone. A specific complex between UDG and duplex DNA containing 2'-beta-fluorodeoxyuridine shows similar increases in the level of DNA base stacking, but also a substrate-directed conformational change in UDG that is not observed with undamaged DNA, consistent with an induced-fit mechanism for damage site recognition. The similar increases in the level of DNA base stacking for the nonspecific and specific complexes suggest a common enzyme-induced distortion in the DNA, potentially DNA bending. The difference spectrum of the extrahelical uracil base in the substrate-analogue complexes reveals only a small electron density reorganization in the uracil ring for the ground state complex, but large 34 cm(-)(1) downshifts in the carbonyl normal modes. Thus, UDG activates the uracil ring in the ground state mainly through H bonds to its C=O groups, without destroying its quasi-aromaticity. This result is at variance with the conclusion from a recent crystal structure, in which the UDG active site significantly distorts the flipped-out pseudouridine analogue such that a change in hybridization at C1 occurs [Parikh, S. S., et al. (2000) Proc. Natl. Acad. Sci. USA 97, 5083]. The Raman vibrational signature of the bound uracil product differs significantly from that of free uracil at neutral pH, and indicates that the uracil is anionic. This is consistent with recent NMR results, which established that the enzyme stabilizes the uracil anion leaving group by 3.4 pK(a) units compared to aqueous solution, contributing significantly to catalysis. These observations are generally not apparent from the high-resolution crystal structures of UDG and its complexes with DNA; thus, Raman spectroscopy can provide unique and valuable insights into the nature of enzyme-DNA interactions.
利用非共振拉曼光谱，我们研究了DNA修复酶尿嘧啶DNA糖基化酶（UDG）催化途径上的每个复合物。未受损DNA与UDG的结合导致DNA拉曼谱带强度降低，这可归因于碱基堆积水平的提高，而DNA主链的振动模式几乎没有扰动。UDG与含有2'-β-氟脱氧尿苷的双链DNA之间的特定复合物显示出类似的DNA碱基堆积水平增加，但UDG中也存在未受损DNA未观察到的底物导向构象变化，这与损伤位点识别的诱导契合机制一致。非特异性和特异性复合物中DNA碱基堆积水平的类似增加表明，酶诱导了DNA中常见的扭曲，可能是DNA弯曲。底物类似物复合物中螺旋外尿嘧啶碱基的差谱显示，基态复合物中尿嘧啶环仅发生了小的电子密度重新排列，但羰基正常模式有34 cm(-1)的大幅下移。因此，UDG主要通过与尿嘧啶C=O基团形成氢键在基态激活尿嘧啶环，而不破坏其准芳香性。这一结果与最近的晶体结构得出的结论不同，在该晶体结构中，UDG活性位点显著扭曲了翻转出的假尿苷类似物，使得C1处的杂交发生了变化[Parikh, S. S., 等人（2000年）《美国国家科学院院刊》97, 5083]。结合的尿嘧啶产物的拉曼振动特征在中性pH下与游离尿嘧啶有显著差异，表明尿嘧啶是阴离子形式。这与最近的核磁共振结果一致，该结果表明，与水溶液相比，该酶使尿嘧啶阴离子离去基团的pK(a)值稳定了3.4个单位，对催化作用有显著贡献。这些观察结果从UDG及其与DNA复合物的高分辨率晶体结构中通常并不明显；因此，拉曼光谱可以为酶与DNA相互作用的本质提供独特而有价值的见解。
