Lee H W, Kim S, Paik W K
Biochemistry. 1977 Jan 11;16(1):78-85. doi: 10.1021/bi00620a013.
Protein methylase I (S-adenosylmethionine: protein-arginine methyltransferase, EC 2.1.1.23) has been purified from calf brain approximately 120-fold with a 14% yield. The final preparation is completely free of any other protein-specific methyltransferases and endogenous substrate protein. The enzyme has an optimum pH of 7.2 and pI value of 5.1. The Km values for S-adenosyl-L-methionine, histone H4, and an ancephalitogenic basic protein are 7.6 X 10(-6), 2.5 X 10(-5), and 7.1 X 10(-5) M, respectively, and the Ki value for S-adenosyl-L-homocysteine is 2.62 X 10(-6) M. The enzyme is highly specific for the arginine residues of protein, and the end products after hydrolysis of the methylated protein are NG,NG-di(asymmetric), NG,N'G-di(symmetric), and NG-monomethylarginine. The ratio of [14C]methyl incorporation into these derivatives by enzyme preparation at varying stages of purification remains unchanged at 40:5:55, strongly indicating that a single enzyme is involved in the synthesis of the three arginine derivatives. The kinetic mechanism of the protein methylase I reaction was studied with the purified enzyme. Initial velocity patterns converging at a point on the extended axis of abscissas were obtained with either histone H4 or S-adenosyl-L-methionine as the varied substrate. Product inhibition by S-adenosyl-L-homocysteine with S-adenosyl-L-methionine as the varied substrate was competitive regardless of whether or not the enzyme was saturated with histone H4. On the other hand, when histone H4 is the variable substrate, noncompetitive inhibition was obtained with S-adenosyl-L-homocysteine under conditions where the enzyme is not saturated with the other substrate, S-adenosyl-L-methionine. These results suggest that the mechanism of the protein methylase I reaction is a Sequential Ordered Bi Bi mechanism with S-adenosyl-L-methionine as the first substrate, histone H4 as the second substrate, methylated histone H4 as the first product, and S-adenosyl-L-homocysteine as the second product released.
蛋白甲基化酶I（S-腺苷甲硫氨酸：蛋白精氨酸甲基转移酶，EC 2.1.1.23）已从小牛脑中纯化出来，纯化倍数约为120倍，产率为14%。最终制剂完全不含任何其他蛋白特异性甲基转移酶和内源性底物蛋白。该酶的最适pH为7.2，pI值为5.1。S-腺苷-L-甲硫氨酸、组蛋白H4和一种致脑炎性碱性蛋白的Km值分别为7.6×10⁻⁶、2.5×10⁻⁵和7.1×10⁻⁵ M，S-腺苷-L-高半胱氨酸的Ki值为2.62×10⁻⁶ M。该酶对蛋白质的精氨酸残基具有高度特异性，甲基化蛋白质水解后的终产物为NG,NG-二（不对称）、NG,N'G-二（对称）和NG-单甲基精氨酸。在纯化的不同阶段，酶制剂将[¹⁴C]甲基掺入这些衍生物中的比例保持在40:5:55不变，这有力地表明单一酶参与了三种精氨酸衍生物的合成。用纯化的酶研究了蛋白甲基化酶I反应的动力学机制。以组蛋白H4或S-腺苷-L-甲硫氨酸作为变化的底物时，得到了在横坐标延长轴上某一点收敛的初速度模式。以S-腺苷-L-甲硫氨酸作为变化的底物时，S-腺苷-L-高半胱氨酸的产物抑制是竞争性的，无论酶是否被组蛋白H4饱和。另一方面，当组蛋白H4是可变底物时，在酶未被另一种底物S-腺苷-L-甲硫氨酸饱和的条件下，S-腺苷-L-高半胱氨酸产生非竞争性抑制。这些结果表明，蛋白甲基化酶I反应的机制是一种有序的顺序双底物双产物机制，其中S-腺苷-L-甲硫氨酸是第一种底物，组蛋白H4是第二种底物，甲基化的组蛋白H4是第一种产物，S-腺苷-L-高半胱氨酸是释放的第二种产物。
