Phenazine-1-carboxylic acid decarboxylase (PhdA) is a prenylated-FMN-dependent (prFMN) enzyme belonging to the UbiD family of decarboxylases. Many UbiD-like enzymes catalyze (de)carboxylation reactions on aromatic rings and conjugated double bonds and are potentially valuable industrial catalysts. We have investigated the mechanism of PhdA using a slow turnover substrate, 2,3-dimethylquinoxaline-5-carboxylic acid (DQCA). Detailed analysis of the pH dependence and solvent deuterium isotope effects associated with the reaction uncovered unusual kinetic behavior. At low substrate concentrations, a substantial inverse solvent isotope effect (SIE) is observed on V_max/K_M of ∼ 0.5 when reaction rates of DQCA in H₂O and D₂O are compared. Under the same conditions, a normal SIE of 4.15 is measured by internal competition for proton transfer to the product. These apparently contradictory results indicate that the SIE values report on different steps in the mechanism. A proton inventory analysis of the reaction under V_max/K_M and V_max conditions points to a “medium effect” as the source of the inverse SIE. Molecular dynamics simulations of the effect of D₂O on PhdA structure support that D₂O reduces the conformational lability of the enzyme and results in a more compact structure, akin to the active, “closed” conformer observed in crystal structures of some UbiD-like enzymes. Consistent with the simulations, PhdA was found to be more stable in D₂O and to bind DQCA more tightly, leading to the observed rate enhancement under V_max/K_M conditions.

吩嗪-1-羧酸脱羧酶 (PhdA) 是一种异戊二烯化 FMN 依赖性 (prFMN) 酶，属于脱羧酶 UbiD 家族。许多类 UbiD 酶催化芳环和共轭双键上的（脱）羧化反应，是潜在有价值的工业催化剂。我们使用缓慢周转底物 2,3-二甲基喹喔啉-5-羧酸 (DQCA) 研究了 PhdA 的机制。对与反应相关的 pH 依赖性和溶剂氘同位素效应的详细分析揭示了不寻常的动力学行为。在低底物浓度下，当比较DQCA 在 H ₂ O 和 D _{2 O 中的反应速率时，在}V _max /K _M约为 0.5 时观察到显着的反溶剂同位素效应 (SIE)。在相同条件下，通过质子转移到产物的内部竞争测得正常SIE 为 4.15。这些明显矛盾的结果表明 SIE 值报告了机制中的不同步骤。在V _max /K _M和V _max条件下反应的质子库存分析表明“中等效应”是逆SIE的来源。 D ₂ O 对 PhdA 结构影响的分子动力学模拟支持 D ₂ O 降低了酶的构象不稳定性并导致结构更加紧凑，类似于在一些 UbiD-晶体结构中观察到的活性“闭合”构象异构体。像酶一样。与模拟一致，发现 PhdA 在 D ₂ O 中更稳定，并且与 DQCA 结合更紧密，导致在V _max /K _M条件下观察到速率增强。