Kern D, Kern G, Neef H, Tittmann K, Killenberg-Jabs M, Wikner C, Schneider G, Hübner G
Institut für Biochemie, Martin-Luther Universität Halle-Wittenberg, Kurt-Mothes-Strasse 3, D-06120 Halle, Germany.
Science. 1997 Jan 3;275(5296):67-70. doi: 10.1126/science.275.5296.67.
The controversial question of how thiamine diphosphate, the biologically active form of vitamin B1, is activated in different enzymes has been addressed. Activation of the coenzyme was studied by measuring thermodynamics and kinetics of deprotonation at the carbon in the 2-position (C2) of thiamine diphosphate in the enzymes pyruvate decarboxylase and transketolase by use of nuclear magnetic resonance spectroscopy, proton/deuterium exchange, coenzyme analogs, and site-specific mutant enzymes. Interaction of a glutamate with the nitrogen in the 1'-position in the pyrimidine ring activated the 4'-amino group to act as an efficient proton acceptor for the C2 proton. The protein component accelerated the deprotonation of the C2 atom by several orders of magnitude, beyond the rate of the overall enzyme reaction. Therefore, the earlier proposed concerted mechanism or stabilization of a C2 carbanion can be excluded.
维生素B1的生物活性形式——硫胺素二磷酸如何在不同酶中被激活这一颇具争议的问题已得到探讨。通过利用核磁共振光谱、质子/氘交换、辅酶类似物和位点特异性突变酶，对丙酮酸脱羧酶和转酮醇酶中硫胺素二磷酸2位（C2）碳原子去质子化的热力学和动力学进行测量，研究了辅酶的激活过程。谷氨酸与嘧啶环1'-位氮原子的相互作用激活了4'-氨基，使其成为C2质子的有效质子受体。蛋白质组分将C2原子的去质子化加速了几个数量级，超过了整个酶反应的速率。因此，可以排除先前提出的协同机制或C2碳负离子的稳定作用。
