CHEMICAL MODIFICATION OF A FUNCTIONAL ARGININE RESIDUE OF RAT-LIVER GLYCINE METHYLTRANSFERASE
CHEMICAL MODIFICATION OF A FUNCTIONAL ARGININE RESIDUE OF RAT-LIVER GLYCINE METHYLTRANSFERASE
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DOI：
10.1021/bi00399a069
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发表时间：
1987-12-15
期刊：
影响因子：
2.9
通讯作者：
FUJIOKA, M
中科院分区：
文献类型：
--
作者：
KONISHI, K;FUJIOKA, M
磷酸钾缓冲液中苯乙二醛对大鼠肝甘氨酸甲基转移酶的不可逆失活。其失活符合拟一级动力学，失活的表观一级速率常数与试剂浓度呈线性关系。二级速率常数为10.54 ±。0.44在pH 8.2和25 ℃下获得M-1 min-1。C.氨基酸分析表明，只有精氨酸被修饰后处理与苯乙二醛。乙酸钠是甘氨酸的竞争性抑制剂，在S-腺苷甲硫氨酸存在下提供完全保护。单独的醋酸盐对灭活率没有影响。从保护实验中测定的乙酸解离常数的值与动力学分析得到的值吻合得很好。在存在和不存在保护配体的情况下，将[14 C]苯基乙二醛掺入蛋白质中的量和修饰的精氨酸残基的数目进行比较，表明每个酶亚基修饰一个精氨酸残基消除了酶活性，并且通过肽分析将该残基鉴定为Arg-175。甘氨酸修饰的甘氨酸甲基转移酶似乎结合S-腺苷甲硫氨酸的天然酶，从S-腺苷甲硫氨酸的蛋白质荧光淬灭。这些结果表明需要Arg-175结合底物甘氨酸的羧基。
Rat liver glycine methyltransferase is inactivated irreversibly by phenylglyoxal in potassium phosphate buffer. The inactivation obeys pseudo-first-order kinetics, and the apparent first-order rate constant for inactivation is linearly related to the reagent concentration. A second-order rate constant of 10.54 .+-. 0.44 M-1 min-1 is obtained at pH 8.2 and 25.degree. C. Amino acid analysis shows that only arginine is modified upon treatment with phenylglyoxal. Sodium acetate, a competitive inhibitor with respect to glycine, affords complete protection in the presence of S-adenosylmethionine. Acetate alone has no effect on the rate of inactivation. The value of the dissociation constant for acetate determined from the protection experiment is in good agreement with that obtained by kinetic analysis. Comparison of the amount of [14C]phenylglyoxal incorporated into the protein and the number of arginine residues modified in the presence and absence of protecting ligands indicates that modification of one arginine residue per enzyme subunit eliminates the enzyme activity, and this residue is identified as Arg-175 by peptide analysis. The arginine-modified glycine methyltransferase appears to bind S-adenosylmethionine as the native enzyme does, as seen from quenching of the protein fluorescence by S-adenosylmethionine. These results suggest the requirement of Arg-175 in binding the carboxyl group of the substrate glycine.