Podschun B, Jahnke K, Schnackerz K D, Cook P F
Theodor-Boveri-Institut für Biowissenschaften (Biozentrum), Universität Würzburg, Germany.
J Biol Chem. 1993 Feb 15;268(5):3407-13.
Primary deuterium (NADPH(D)), solvent deuterium, and multiple isotope effects and the pH dependence of kinetic parameters have been used to probe the mechanism of the dihydropyrimidine dehydrogenase from pig liver. Isotope effect and pH-rate data suggest a rate-determining reductive half-reaction in which reduction of the flavin by NADPH has only a minor rate limitation (DV approximately D(V/KNADPH) approximately 1.1), while protonation of the flavin at N-1 occurring in a step following reduction is slow (D2OV = 3, while D2O(V/KNADPH) = 2). An enzymatic general acid with a pK of 8.2 is required to protonate N-1 of the flavin. In the second half-reaction, uracil is reduced at C-6 by flavin and protonated on the opposite face at C-5 by an enzymatic general acid with a pK of 9. The hydride transfer from N-5 of the flavin to C-5 of uracil is facilitated by an enzymatic general base with a pK of 5.6 that accepts a proton from N-1 of the flavin. There is also evidence from the pH dependence of V and the V/K for reduced dinucleotide substrates that a second enzyme residue with a pK of 6.4 must be unprotonated for optimum activity, but is not essential for activity. None of the functional groups reflected in the V/KNADPH pH-rate profile have a role in binding, while both of those observed in the V/Kuracil profile have a role in binding as shown by the pH dependence of the dissociation constants for the competitive inhibitors ATP-ribose and 2,6-dihydroxypyridine.
利用初级氘（NADPH(D)）、溶剂氘、多重同位素效应以及动力学参数对pH的依赖性，来探究猪肝中二氢嘧啶脱氢酶的作用机制。同位素效应和pH-速率数据表明，存在一个速率决定的还原半反应，其中NADPH对黄素的还原仅具有较小的速率限制（DV约等于D(V/KNADPH)约等于1.1），而在还原后的步骤中黄素在N-1处的质子化较慢（D2OV = 3，而D2O(V/KNADPH) = 2）。需要一个pK为8.2的酶促广义酸来使黄素的N-1质子化。在第二个半反应中，尿嘧啶在C-6处被黄素还原，并在相反的C-5面上被一个pK为9的酶促广义酸质子化。黄素N-5上的氢化物向尿嘧啶C-5的转移由一个pK为5.6的酶促广义碱促进，该碱从黄素的N-1接受一个质子。从V和还原二核苷酸底物的V/K对pH的依赖性也有证据表明，一个pK为6.4的第二个酶残基必须去质子化才能达到最佳活性，但对活性不是必需的。V/KNADPH pH-速率曲线中反映的功能基团在结合中均无作用，而在V/K尿嘧啶曲线中观察到的两个功能基团在结合中均有作用，这由竞争性抑制剂ATP-核糖和2,6-二羟基吡啶的解离常数对pH的依赖性所示。
