Jamaluddin M, Kim S, Paik W K
Biochemistry. 1976 Jul 13;15(14):3077-81. doi: 10.1021/bi00659a022.
Kinetic properties of protein methylase II (S-adenosymethionine:protein O-methyltransferase, EC 2.1.1.24) which methylates (esterifies) the free carboxyl side chains of amino acids in proteins was studied using various polypeptides as methyl acceptor substrates. Bovine pancreatic ribonuclease, a model substrate for the enzyme, was subjected to specific cleavage by cyanogen bromide, trypsin, and performic acid oxidation. Several polypeptide fragments derived were then separated by molecular sieve chromatography on a column of Sephadex G-25. The method was found to be very simple and gave good yields. Km values for these polypeptides as well as a few other protein substrates were determined. While Km values for the isolated peptides range generally between 4.8 and 0.7 X 10-3 M, those of native bovine panreatic ribonuclease, luteinizing hormone, and follicle-stimulating hormone were determined to be 4.0 X 10-4, 5.0 X 10-5, and 0.77 X 10-5, respectively. Sites of enzymatic methylation of the native ribonuclease were also investigated. Although polypeptides derived from the C-terminal and N-terminal regions of the molecule were found to accept methyl groups, they were unable to under go enzymatic methylation when native molecule was used as the substrate indicating that within the native ribonuclease these regions are in a conformation which do not allow them to be methylated by protein methylase II under the present assay conditions.
利用各种多肽作为甲基受体底物，对蛋白质甲基化酶II（S-腺苷甲硫氨酸：蛋白质O-甲基转移酶，EC 2.1.1.24）的动力学性质进行了研究，该酶可使蛋白质中氨基酸的游离羧基侧链甲基化（酯化）。牛胰核糖核酸酶作为该酶的模型底物，经溴化氰、胰蛋白酶和过甲酸氧化进行特异性切割。然后通过在Sephadex G-25柱上进行分子筛色谱分离得到几个衍生的多肽片段。该方法非常简单且产率良好。测定了这些多肽以及其他一些蛋白质底物的Km值。分离出的肽段的Km值一般在4.8至0.7×10⁻³M之间，而天然牛胰核糖核酸酶、促黄体生成素和促卵泡激素的Km值分别测定为4.0×10⁻⁴、5.0×10⁻⁵和0.77×10⁻⁵。还研究了天然核糖核酸酶的酶促甲基化位点。虽然发现从分子的C末端和N末端区域衍生的多肽能够接受甲基，但当以天然分子作为底物时，它们无法进行酶促甲基化，这表明在天然核糖核酸酶中，这些区域处于一种构象，在当前的测定条件下不允许它们被蛋白质甲基化酶II甲基化。
