Effects of mutations at tyrosine 66 and asparagine 123 in the active site pocket of Escherichia coli uracil DNA glycosylase on uracil excision from synthetic DNA oligomers:: evidence for the occurrence of long-range interactions between the enzyme and substrate
Effects of mutations at tyrosine 66 and asparagine 123 in the active site pocket of Escherichia coli uracil DNA glycosylase on uracil excision from synthetic DNA oligomers:: evidence for the occurrence of long-range interactions between the enzyme and substrate
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DOI：
10.1093/nar/gkf425
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发表时间：
2002-07-15
影响因子：
14.9
通讯作者：
Varshney, U
中科院分区：
文献类型：
--
作者：
Handa, P;Acharya, N;Varshney, U
尿嘧啶DNA糖基化酶（UDG）是一种高度保守的DNA修复酶，从DNA中切除尿嘧啶。几种UDG的晶体结构已经鉴定出对于其在检测和去除尿嘧啶中的精确特异性重要的残基。其中，已经提出大肠杆菌UDG中的Y 66和N123限制非尿嘧啶残基进入活性位点口袋。在这项研究中，我们发现Y 66 F突变体的尿嘧啶切除活性与野生型蛋白相似，而其他突变体（Y 66 C，Y 66 S，N123 D，N123 E和N123 Q）的活性降低了1000倍。后一类的突变体表现出增加的依赖于底物链的长度，并建议存在的远程相互作用的基板与UDG。通过乙基化干扰试验对磷酸盐相互作用的研究再次证实了-1、+1和+2磷酸盐（相对于易断裂的尿嘧啶）对酶活性的关键重要性。有趣的是，该测定还揭示了-5位磷酸的额外干扰，其在底物中的存在对活性位点口袋中不具有完全互补相互作用的突变体的底物利用具有积极影响。这种长程相互作用可能是至关重要的，即使是野生型酶在体内条件下。此外，我们的研究结果表明，Y 66和N123在UDG中的作用不仅限于防止非尿嘧啶残基的进入。我们讨论了它们在赋予过渡态酶-底物复合物稳定性和/或增强催化过程中尿嘧啶酸阴离子离去基团质量方面的额外作用。
Uracil DNA glycosylase (UDG), a highly conserved DNA repair enzyme, excises uracil from DNA. Crystal structures of several UDGs have identified residues important for their exquisite specificity in detection and removal of uracil. Of these, Y66 and N123 in Escherichia coli UDG have been proposed to restrict the entry of non-uracil residues into the active site pocket. In this study, we show that the uracil excision activity of the Y66F mutant was similar to that of the wild-type protein, whereas the activities of the other mutants (Y66C, Y66S, N123D, N123E and N123Q) were compromised similar to1000-fold. The latter class of mutants showed an increased dependence on the substrate chain length and suggested the existence of long-range interactions of the substrate with UDG. Investigation of the phosphate interactions by the ethylation interference assay reaffirmed the key importance of the -1, +1 and +2 phosphates (with respect to the scissile uracil) to the enzyme activity. Interestingly, this assay also revealed an additional interference at the -5 position phosphate, whose presence in the substrate had a positive effect on substrate utilisation by the mutants that do not possess a full complement of interactions in the active site pocket. Such long-range interactions may be crucial even for the wild-type enzyme under in vivo conditions. Further, our results suggest that the role of Y66 and N123 in UDG is not restricted merely to preventing the entry of non-uracil residues. We discuss their additional roles in conferring stability to the transition state enzyme-substrate complex and/or enhancing the leaving group quality of the uracilate anion during catalysis.