Wyszynski M W, Gabbara S, Kubareva E A, Romanova E A, Oretskaya T S, Gromova E S, Shabarova Z A, Bhagwat A S
Department of Chemistry, Wayne State University, Detroit, MI 48202.
Nucleic Acids Res. 1993 Jan 25;21(2):295-301. doi: 10.1093/nar/21.2.295.
All DNA (cytosine-5)-methyltransferases contain a single conserved cysteine. It has been proposed that this cysteine initiates catalysis by attacking the C6 of cytosine and thereby activating the normally inert C5 position. We show here that substitutions of this cysteine in the E. coli methylase M. EcoRII with either serine or tryptophan results in a complete loss of ability to transfer methyl groups to DNA. Interestingly, mutants with either serine or glycine substitution bind tightly to substrate DNA. These mutants resemble the wild-type enzyme in that their binding to substrate is not eliminated by the presence of non-specific DNA in the reaction, it is sensitive to methylation status of the substrate and is stimulated by an analog of the methyl donor. Hence the conserved cysteine is not essential for the specific stable binding of the enzyme to its substrate. However, substitution of the cysteine with the bulkier tryptophan does reduce DNA binding. We also report here a novel procedure for the synthesis of DNA containing 5-fluorocytosine. Further, we show that a DNA substrate for M. EcoRII in which the target cytosine is replaced by 5-fluorocytosine is a mechanism-based inhibitor of the enzyme and that it forms an irreversible complex with the enzyme. As expected, this modified substrate does not form irreversible complexes with the mutants.
所有DNA（胞嘧啶-5）-甲基转移酶都含有一个保守的半胱氨酸。有人提出，这个半胱氨酸通过攻击胞嘧啶的C6来启动催化作用，从而激活通常惰性的C5位置。我们在此表明，用丝氨酸或色氨酸替换大肠杆菌甲基化酶M. EcoRII中的这个半胱氨酸会导致其将甲基转移到DNA上的能力完全丧失。有趣的是，丝氨酸或甘氨酸替换的突变体与底物DNA紧密结合。这些突变体与野生型酶相似，它们与底物的结合不会因反应中存在非特异性DNA而消除，对底物的甲基化状态敏感，并受到甲基供体类似物的刺激。因此，保守的半胱氨酸对于酶与其底物的特异性稳定结合并非必不可少。然而，用体积更大的色氨酸替换半胱氨酸确实会降低DNA结合。我们在此还报告了一种合成含5-氟胞嘧啶DNA的新方法。此外，我们表明，M. EcoRII的一种DNA底物，其中靶胞嘧啶被5-氟胞嘧啶取代，是该酶的一种基于机制的抑制剂，并且它与该酶形成不可逆复合物。正如预期的那样，这种修饰的底物不会与突变体形成不可逆复合物。
