Department of Chemistry, University of Michigan, Ann Arbor, Michigan, USA.
Department of Chemical Engineering, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA.
J Biol Chem. 2024 Feb;300(2):105621. doi: 10.1016/j.jbc.2023.105621. Epub 2024 Jan 3.
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/K of ∼ 0.5 when reaction rates of DQCA in HO and DO 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/K and V conditions points to a "medium effect" as the source of the inverse SIE. Molecular dynamics simulations of the effect of DO on PhdA structure support that DO 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 DO and to bind DQCA more tightly, leading to the observed rate enhancement under V/K conditions.
苯并嗪-1-羧酸脱羧酶（PhdA）是一种 prenylated-FMN 依赖性（prFMN）酶，属于 UbiD 家族的脱羧酶。许多 UbiD 样酶催化芳香环和共轭双键上的（脱）羧化反应，是潜在有价值的工业催化剂。我们使用低周转率的底物 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异常的动力学行为。在低底物浓度下，当比较 HO 和 DO 中 DQCA 的反应速率时，观察到 V/K 的显著逆溶剂同位素效应（SIE）约为 0.5。在相同条件下，通过产物质子转移的内部竞争，测量到正常的 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/K 和 V 条件下对反应的质子库存分析表明，“介质效应”是逆 SIE 的来源。对 DO 对 PhdA 结构影响的分子动力学模拟支持 DO 降低了酶的构象不稳定性，并导致更紧凑的结构，类似于某些 UbiD 样酶的晶体结构中观察到的活性“封闭”构象。与模拟一致，PhdA 在 DO 中更稳定，并且与 DQCA 的结合更紧密，导致在 V/K 条件下观察到的速率增强。
