Reich N O, Maegley K A, Shoemaker D D, Everett E
Department of Chemistry, University of California, Santa Barbara 93106.
Biochemistry. 1991 Mar 19;30(11):2940-6. doi: 10.1021/bi00225a030.
Native EcoRI DNA methyltransferase (Mtase, Mr 38,050) is proteolyzed by trypsin to generate an intermediate 36-kDa fragment (p36) followed by the formation of two polypeptides of Mr 23,000 and 13,000 (p23 and p13, respectively). Protein sequence analysis of the tryptic fragments indicates that p36 results from removal of the first 14 or 16 amino acids, p23 spans residues 15-216, and p13 spans residues 217-325. The relative resistance to further degradation of p23 and p13 suggests stable domain structures. This is further supported by the generation of similar fragments with SV8 endoprotease which has entirely different peptide specificities. Our results suggest the Mtase is a two-domain protein connected by a highly flexible interdomain hinge. The putative hinge region encompasses previously identified peptides implicated in AdoMet binding [Reich, N.O., & Everett, E. (1990) J. Biol. Chem. 265, 8929-8934] and catalysis [Everett et al. (1990) J. Biol. Chem. 265, 17713-17719]. Protection studies with DNA, S-adenosylmethionine (AdoMet), S-adenosylhomocysteine (AdoHcy), and sinefungin (AdoMet analogue) show that the Mtase undergoes significant conformational changes upon ligand binding. Trypsinolysis of the AdoMet-bound form of the Mtase generates different fragments, and the AdoMet-bound form is over 800 times more stable than unbound Mtase. The sequence-specific ternary complex (Mtase-DNA-sinefungin) is 2000 times more resistant to degradation by trypsin; cleavage eventually generates 26- and 12-kDa fragments which span residues 104-325 and 1-103, respectively (p26 and p12). The first 14 or 16 amino acids of the Mtase are not essential since p36 retains activity. Activity analysis of the p26 and p12 mixture also indicates retention of activity.(ABSTRACT TRUNCATED AT 250 WORDS)
天然的 EcoRI DNA 甲基转移酶（Mtase，分子量 38,050）被胰蛋白酶水解，产生一个 36 kDa 的中间片段（p36），随后形成分子量分别为 23,000 和 13,000 的两种多肽（分别为 p23 和 p13）。对胰蛋白酶片段的蛋白质序列分析表明，p36 是由于去除了前 14 或 16 个氨基酸产生的，p23 涵盖第 15 - 216 位残基，p13 涵盖第 217 - 325 位残基。p23 和 p13 对进一步降解的相对抗性表明其结构域结构稳定。具有完全不同肽特异性的 SV8 内切蛋白酶产生类似片段进一步支持了这一点。我们的结果表明，Mtase 是一种由高度灵活的结构域间铰链连接的双结构域蛋白。推测的铰链区包含先前鉴定的与 AdoMet 结合[Reich, N.O., & Everett, E. (1990) J. Biol. Chem. 265, 8929 - 8934]和催化作用[Everett 等人 (1990) J. Biol. Chem. 265, 17713 - 17719]相关的肽段。用 DNA、S - 腺苷甲硫氨酸（AdoMet）、S - 腺苷高半胱氨酸（AdoHcy）和杀稻瘟菌素（AdoMet 类似物）进行的保护研究表明，Mtase 在配体结合时会发生显著的构象变化。对 Mtase 的 AdoMet 结合形式进行胰蛋白酶消化会产生不同的片段，并且 AdoMet 结合形式比未结合的 Mtase 稳定 800 多倍。序列特异性三元复合物（Mtase - DNA - 杀稻瘟菌素）对胰蛋白酶降解的抗性高 2000 倍；切割最终产生 26 kDa 和 12 kDa 的片段，分别涵盖第 104 - 325 位残基和第 1 - 103 位残基（p26 和 p12）。Mtase 的前 14 或 16 个氨基酸不是必需的，因为 p36 保留了活性。p26 和 p12 混合物的活性分析也表明活性得以保留。（摘要截短于 250 字）
