Bennett Matthew T, Rodgers M T, Hebert Alexander S, Ruslander Lindsay E, Eisele Leslie, Drohat Alexander C
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
J Am Chem Soc. 2006 Sep 27;128(38):12510-9. doi: 10.1021/ja0634829.
Initiating the DNA base excision repair pathway, DNA glycosylases find and hydrolytically excise damaged bases from DNA. While some DNA glycosylases exhibit narrow specificity, others remove multiple forms of damage. Human thymine DNA glycosylase (hTDG) cleaves thymine from mutagenic G.T mispairs, recognizes many additional lesions, and has a strong preference for nucleobases paired with guanine rather than adenine. Yet, hTDG avoids cytosine, despite the million-fold excess of normal G.C pairs over G.T mispairs. The mechanism of this remarkable and essential specificity has remained obscure. Here, we examine the possibility that hTDG specificity depends on the stability of the scissile base-sugar bond by determining the maximal activity (k(max)) against a series of nucleobases with varying leaving-group ability. We find that hTDG removes 5-fluorouracil 78-fold faster than uracil, and 5-chlorouracil, 572-fold faster than thymine, differences that can be attributed predominantly to leaving-group ability. Moreover, hTDG readily excises cytosine analogues with improved leaving ability, including 5-fluorocytosine, 5-bromocytosine, and 5-hydroxycytosine, indicating that cytosine has access to the active site. A plot of log(k(max)) versus leaving-group pK(a) reveals a Brønsted-type linear free energy relationship with a large negative slope of beta(lg) = -1.6 +/- 0.2, consistent with a highly dissociative reaction mechanism. Further, we find that the hydrophobic active site of hTDG contributes to its specificity by enhancing the inherent differences in substrate reactivity. Thus, hTDG specificity depends on N-glycosidic bond stability, and the discrimination against cytosine is due largely to its very poor leaving ability rather than its exclusion from the active site.
启动DNA碱基切除修复途径时，DNA糖基化酶会找到并通过水解作用从DNA中切除受损碱基。虽然一些DNA糖基化酶表现出狭窄的特异性，但其他酶能去除多种形式的损伤。人类胸腺嘧啶DNA糖基化酶（hTDG）从诱变的G.T错配中切割胸腺嘧啶，识别许多其他损伤，并且对与鸟嘌呤而非腺嘌呤配对的核碱基有强烈偏好。然而，尽管正常的G.C对与G.T错配的比例高达百万倍，hTDG仍避开胞嘧啶。这种显著且至关重要的特异性机制一直不清楚。在这里，我们通过测定hTDG对一系列具有不同离去基团能力的核碱基的最大活性（k(max)），来研究hTDG特异性是否取决于可裂解碱基 - 糖键的稳定性。我们发现hTDG切除5 - 氟尿嘧啶的速度比尿嘧啶快78倍，切除5 - 氯尿嘧啶的速度比胸腺嘧啶快572倍，这些差异主要可归因于离去基团的能力。此外，hTDG能轻易切除离去能力增强的胞嘧啶类似物，包括5 - 氟胞嘧啶、5 - 溴胞嘧啶和5 - 羟基胞嘧啶，这表明胞嘧啶能够进入活性位点。log(k(max))与离去基团pK(a)的关系图显示出一种布朗斯特型线性自由能关系，其大的负斜率β(lg) = -1.6 +/- 0.2，这与高度解离的反应机制一致。此外，我们发现hTDG的疏水活性位点通过增强底物反应性的固有差异来促进其特异性。因此，hTDG的特异性取决于N - 糖苷键的稳定性，对胞嘧啶的歧视主要是由于其极差的离去能力，而非被排除在活性位点之外。
