McArdell J E, Bruton C J, Atkinson T
Public Health Laboratory Service, Centre for Applied Microbiology and Research, Porton Down, Wilts, U.K.
Biochem J. 1987 May 1;243(3):701-7. doi: 10.1042/bj2430701.
Tryptophyl-tRNA synthetase is irreversibly inactivated by Procion Brown MX-5BR with an apparent dissociation constant (KD) of 8.8 microM and maximum rate of inactivation k3 0.192 s-1. The specificity of the interaction is supported by two previously reported observations. Firstly, Brown MX-5BR inactivation of tryptophyl-tRNA synthetase is inhibited by substrates, and secondly, the animated derivative of Brown MX-5BR is a competitive inhibitor of tryptophyl-tRNA synthetase with a Ki of 2 X 10(-4) M with respect to both tryptophan and ATP. Tryptic digestion of the dye-affinity-labelled enzyme and subsequent resolution of the peptides by h.p.l.c. yielded one major dye-peptide peak. Amino acid sequence analysis resulted in the identification of the dye-binding domain centred on lysine-178. Tyrosyl-tRNA synthetase is also inactivated by Procion Brown MX-5BR, and this inactivation is prevented by ATP but not by tyrosine. The interaction of tyrosyl-tRNA synthetase with hydroxylated Brown MX-5BR exhibited non-competitive kinetics with respect to the amino acid-binding site and competitive kinetics against ATP with a Ki of 6 X 10(-6) M.
色氨酰 - tRNA合成酶被普施安棕MX - 5BR不可逆地失活，其表观解离常数（KD）为8.8微摩尔，最大失活速率k3为0.192秒-1。先前报道的两个观察结果支持了这种相互作用的特异性。首先，色氨酰 - tRNA合成酶的普施安棕MX - 5BR失活受到底物的抑制，其次，普施安棕MX - 5BR的胺化衍生物是色氨酰 - tRNA合成酶的竞争性抑制剂，相对于色氨酸和ATP的Ki为2×10（-4）M。对染料亲和标记的酶进行胰蛋白酶消化，随后通过高效液相色谱法分离肽段，得到一个主要的染料 - 肽峰。氨基酸序列分析确定了以赖氨酸 - 178为中心的染料结合结构域。酪氨酰 - tRNA合成酶也被普施安棕MX - 5BR失活，这种失活被ATP阻止，但不被酪氨酸阻止。酪氨酰 - tRNA合成酶与羟基化的普施安棕MX - 5BR的相互作用在氨基酸结合位点方面表现出非竞争性动力学，对ATP表现出竞争性动力学，Ki为6×10（-6）M。
