Konishi K, Fujioka M
Department of Biochemistry, Toyama Medical and Pharmaceutical University, Faculty of Medicine, Japan.
Biochemistry. 1987 Dec 15;26(25):8496-502. doi: 10.1021/bi00399a069.
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 degrees 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.
在磷酸钾缓冲液中，苯乙二醛可使大鼠肝脏甘氨酸甲基转移酶发生不可逆失活。失活过程遵循假一级动力学，失活的表观一级速率常数与试剂浓度呈线性关系。在pH 8.2和25℃条件下，得到的二级速率常数为10.54±0.44 M-1 min-1。氨基酸分析表明，用苯乙二醛处理后只有精氨酸被修饰。乙酸钠是甘氨酸的竞争性抑制剂，在S-腺苷甲硫氨酸存在时可提供完全保护。单独的乙酸盐对失活速率没有影响。通过保护实验确定的乙酸盐解离常数的值与通过动力学分析得到的值吻合良好。比较在有和没有保护配体存在的情况下，掺入蛋白质中的[14C]苯乙二醛的量以及修饰的精氨酸残基的数量，表明每个酶亚基中一个精氨酸残基的修饰会消除酶活性，通过肽分析确定该残基为Arg-175。从S-腺苷甲硫氨酸对蛋白质荧光的淬灭可以看出，精氨酸修饰的甘氨酸甲基转移酶似乎像天然酶一样结合S-腺苷甲硫氨酸。这些结果表明Arg-175在结合底物甘氨酸的羧基方面的必要性。
