Hübner G, Tittmann K, Killenberg-Jabs M, Schäffner J, Spinka M, Neef H, Kern D, Kern G, Schneider G, Wikner C, Ghisla S
Institut für Biochemie, Martin-Luther-Universität Halle-Wittenberg, Kurt Mothes Str. 3, D-06120 Halle, Germany.
Biochim Biophys Acta. 1998 Jun 29;1385(2):221-8. doi: 10.1016/s0167-4838(98)00070-3.
Activation of the coenzyme ThDP was studied by measuring the kinetics of deprotonation at the C2 carbon of thiamin diphosphate in the enzymes pyruvate decarboxylase, transketolase, pyruvate dehydrogenase complex, pyruvate oxidase, in site-specific mutant enzymes and in enzyme complexes containing coenzyme analogues by proton/deuterium exchange detected by 1H-NMR spectroscopy. The respective deprotonation rate constant is above the catalytic constant in all enzymes investigated. The fast deprotonation requires the presence of an activator in pyruvate decarboxylase from yeast, showing the allosteric regulation of this enzyme to be accomplished by an increase in the C2-H dissociation rate of the enzyme-bound thiamin diphosphate. The data of the thiamin diphosphate analogues and of the mutant enzymes show the N1' atom and the 4'-NH2 group to be essential for the activation of the coenzyme and a conserved glutamate involved in the proton abstraction mechanism of the enzyme-bound thiamin diphosphate.
通过1H-NMR光谱检测质子/氘交换，在丙酮酸脱羧酶、转酮醇酶、丙酮酸脱氢酶复合体、丙酮酸氧化酶、位点特异性突变酶以及含有辅酶类似物的酶复合体中，测量硫胺素二磷酸C2位碳的去质子化动力学，研究辅酶硫胺素焦磷酸（ThDP）的激活情况。在所研究的所有酶中，各自的去质子化速率常数均高于催化常数。快速去质子化需要来自酵母的丙酮酸脱羧酶中存在激活剂，这表明该酶的变构调节是通过酶结合的硫胺素二磷酸的C2-H解离速率增加来实现的。硫胺素二磷酸类似物和突变酶的数据表明，N1'原子和4'-NH2基团对于辅酶的激活至关重要，并且一个保守的谷氨酸参与了酶结合的硫胺素二磷酸的质子提取机制。
