Som S, Friedman S
Department of Pharmacology, State University of New York Health Science Center, Brooklyn 11203.
J Biol Chem. 1990 Mar 15;265(8):4278-83.
Ultraviolet irradiation of EcoRII methyltransferase in the presence of its substrate, S-adenosyl-L-methionine (AdoMet), results in the formation of a stable enzyme-substrate adduct. This adduct can be demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after irradiation of the enzyme in the presence of either [methyl-3H]AdoMet or [35S]AdoMet. The extent of photolabeling is low. Under optimal conditions, 4.5 pmol of [3H]AdoMet is incorporated into 100 pmol of enzyme. Use of the 8-azido derivative of AdoMet as the photolabeling substrate increases the incorporation by approximately 2-fold. However, this adduct, unlike the one formed with AdoMet, is not stable when treated with thiol reagents or precipitated with trichloroacetic acid. A catalytically active conformation of the enzyme is needed for AdoMet photolabeling. Heat-inactivated enzyme or proteins for which AdoMet is not a substrate or cofactor do not undergo adduct formation. Two other methyltransferases, MspI and dam methylases are also shown to form adducts with AdoMet upon UV irradiation. The binding constant of the EcoRII methyltransferase for AdoMet determined with the photolabeling reaction is 11 microM, which is similar to the binding constant of 9 microM previously reported (Friedman, S. (1986) Nucleic Acids Res. 14, 4543-4556). The AdoMet analogs S-adenosyl-L-homocysteine (Ki = 0.83 microM) and sinefungin (Ki = 4.3 microM) are effective inhibitors of photolabeling, whereas S-adenosyl-D-homocysteine (Ki = 46 microM) is a poor inhibitor. These experiments indicate that AdoMet becomes covalently bound at the AdoMet-binding site on the enzyme molecule. The EcoRII methyltransferase-AdoMet adduct is very stable and could be used to identify the AdoMet-binding site on DNA methyltransferases.
在其底物S-腺苷-L-甲硫氨酸（AdoMet）存在的情况下，对EcoRII甲基转移酶进行紫外线照射，会导致形成一种稳定的酶-底物加合物。在[甲基-³H]AdoMet或[³⁵S]AdoMet存在的情况下对该酶进行照射后，这种加合物可以通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳来证明。光标记的程度较低。在最佳条件下，4.5皮摩尔的[³H]AdoMet被掺入100皮摩尔的酶中。使用AdoMet的8-叠氮基衍生物作为光标记底物可使掺入量增加约2倍。然而，与用AdoMet形成的加合物不同，这种加合物在用硫醇试剂处理或用三氯乙酸沉淀时不稳定。AdoMet光标记需要酶的催化活性构象。热失活的酶或AdoMet不是其底物或辅因子的蛋白质不会形成加合物。另外两种甲基转移酶，MspI和dam甲基转移酶，在紫外线照射下也显示会与AdoMet形成加合物。通过光标记反应测定的EcoRII甲基转移酶对AdoMet的结合常数为11微摩尔，这与先前报道的9微摩尔的结合常数相似（弗里德曼，S.（1986年）《核酸研究》14，4543 - 4556）。AdoMet类似物S-腺苷-L-高半胱氨酸（Ki = 0.83微摩尔）和杀稻瘟菌素（Ki = 4.3微摩尔）是光标记的有效抑制剂，而S-腺苷-D-高半胱氨酸（Ki = 46微摩尔）是一种较差的抑制剂。这些实验表明AdoMet在酶分子上的AdoMet结合位点处共价结合。EcoRII甲基转移酶 - AdoMet加合物非常稳定，可用于鉴定DNA甲基转移酶上的AdoMet结合位点。
