Desmadril M, Minard P, Ballery N, Gaillard-Miran S, Hall L, Yon J M
Laboratorie d'Enzymologie physico-chimique et moléculaire, Groupe de Recherche du Centre National de la Recherche Scientifique associé à l'Université de Paris-Sud, Orsay, France.
Proteins. 1991;10(4):315-24. doi: 10.1002/prot.340100405.
The effects of ligands on the conformation of yeast phosphoglycerate kinase were explored by introducing cysteinyl residues at different positions in the molecule by site-directed mutagenesis. Thus several mutants were constructed, each containing a unique cysteinyl residue. Neither the conformation nor the enzyme activity was affected by the substitutions. The reactivity of the thiol groups and the fluorescence of N-acetyl-N'-(5-sulfo-1-naphthyl)ethylene-diamine covalently linked to these thiols were used to monitor the conformational changes induced upon ligand binding. It was found that the observed changes mainly involve the part of the protein located in the cleft, particularly the environment of residues 35 and 183. No alteration was observed on the external side of the protein. Only 3-Phosphoglycerate induced these conformational changes. However, when the fluorescent probe was attached to residue 377, the binding of the two substrates was required to induce a modification in the fluorescence of the probe. These results indicate that the substrates separately or together induce discrete molecular motions in phosphoglycerate kinase.
通过定点诱变在酵母磷酸甘油酸激酶分子的不同位置引入半胱氨酰残基，研究了配体对其构象的影响。由此构建了几个突变体，每个突变体都含有一个独特的半胱氨酰残基。这些取代对构象和酶活性均无影响。利用巯基的反应性以及与这些巯基共价连接的N-乙酰-N'-(5-磺基-1-萘基)乙二胺的荧光来监测配体结合时诱导的构象变化。发现观察到的变化主要涉及位于裂隙中的蛋白质部分，特别是残基35和183周围的环境。在蛋白质外侧未观察到改变。只有3-磷酸甘油酸能诱导这些构象变化。然而，当荧光探针连接到残基377时，需要两种底物结合才能诱导探针荧光发生变化。这些结果表明，底物单独或共同在磷酸甘油酸激酶中诱导离散的分子运动。
