Moe J G, Piszkiewicz D
Biochemistry. 1979 Jun 26;18(13):2810-4. doi: 10.1021/bi00580a019.
The inhibitory effects of blue dextran and a small dye molecule derived from it (F3GA-OH) on the steady-state reaction catalyzed by Escherichia coli isoleucy-tRNA synthetase have been studied. Blue dextran gave uncompetitive inhibition with respect to Mg.ATP, mixed inhibition with respect to L-isoleucine, and competitive inhibition with respect to tRNA. The small dye molecule (F3GA-OH) was also competitive with respect to tRNA. These inhibition patterns were not consistent with the bi-uni-uni-bi Ping Pong mechanism generally accepted for aminoacyl-tRNA synthetases. They were consistent with a mechanism in which a second L-isoleucine is bound after isoleucyl-AMP synthesis and before transfer of the isoleucyl moiety to tRNA. Enzyme-bound L-isoleucine lowered the affinity of the enzyme for blue dextran approximately fivefold, a value comparable to the ninefold lowering of the enzyme's affinity for tRNA upon binding L-isoleucine. The affinity of the synthetase for F3GA-OH (K1 = 1.0 X 10(-7) M) is approximately fivefold higher than its affinity for blue dextran (K1 = 5.3 X 10(-7) M). These results indicate that blue dextran and its derivatives may be useful for kinetic and physical studies of polynucleotide binding sites on proteins as well as NAD and ATP sites.
研究了蓝色葡聚糖及其衍生的一种小染料分子（F3GA - OH）对大肠杆菌异亮氨酰 - tRNA合成酶催化的稳态反应的抑制作用。蓝色葡聚糖对Mg.ATP表现出反竞争性抑制，对L - 异亮氨酸表现出混合型抑制，对tRNA表现出竞争性抑制。小染料分子（F3GA - OH）对tRNA也表现出竞争性抑制。这些抑制模式与通常认为的氨酰 - tRNA合成酶的双 - 单 - 单 - 双乒乓机制不一致。它们与一种机制相符，即在异亮氨酰 - AMP合成后且异亮氨酰部分转移到tRNA之前，有第二个L - 异亮氨酸结合。酶结合的L - 异亮氨酸使酶对蓝色葡聚糖的亲和力降低约五倍，这一数值与结合L - 异亮氨酸后酶对tRNA亲和力降低九倍相当。合成酶对F3GA - OH的亲和力（K1 = 1.0×10⁻⁷ M）比对蓝色葡聚糖的亲和力（K1 = 5.3×10⁻⁷ M）高约五倍。这些结果表明，蓝色葡聚糖及其衍生物可能有助于对蛋白质上的多核苷酸结合位点以及NAD和ATP位点进行动力学和物理研究。
