Tittmann Kai, Golbik Ralph, Uhlemann Kathrin, Khailova Ludmila, Schneider Gunter, Patel Mulchand, Jordan Frank, Chipman David M, Duggleby Ronald G, Hübner Gerhard
Martin-Luther-Universität Halle-Wittenberg, Institut für Biochemie, Kurt-Mothes-Strasse 3, D-06099 Halle, Germany.
Biochemistry. 2003 Jul 8;42(26):7885-91. doi: 10.1021/bi034465o.
Enzymic catalysis proceeds via intermediates formed in the course of substrate conversion. Here, we directly detect key intermediates in thiamin diphosphate (ThDP)-dependent enzymes during catalysis using (1)H NMR spectroscopy. The quantitative analysis of the relative intermediate concentrations allows the determination of the microscopic rate constants of individual catalytic steps. As demonstrated for pyruvate decarboxylase (PDC), this method, in combination with site-directed mutagenesis, enables the assignment of individual side chains to single steps in catalysis. In PDC, two independent proton relay systems and the stereochemical control of the enzymic environment account for proficient catalysis proceeding via intermediates at carbon 2 of the enzyme-bound cofactor. The application of this method to other ThDP-dependent enzymes provides insight into their specific chemical pathways.
酶催化通过底物转化过程中形成的中间体进行。在这里，我们使用¹H NMR光谱法在催化过程中直接检测硫胺素二磷酸（ThDP）依赖性酶中的关键中间体。对相对中间体浓度的定量分析允许确定各个催化步骤的微观速率常数。正如丙酮酸脱羧酶（PDC）所证明的那样，该方法与定点诱变相结合，能够将单个侧链分配到催化中的单个步骤。在PDC中，两个独立的质子传递系统和酶环境的立体化学控制解释了通过酶结合辅因子的C2位中间体进行的高效催化。将该方法应用于其他ThDP依赖性酶可深入了解其特定的化学途径。
