PDF(Pubmed)
Abstract：
Enzymes are potent catalysts that increase biochemical reaction rates by several orders of magnitude. Flavoproteins are a class of enzymes whose classification relies on their ability to react with molecular oxygen (O2) during catalysis using ionizable active site residues. Pseudomonas aeruginosa D-arginine dehydrogenase (PaDADH) is a flavoprotein that oxidizes D-arginine for P. aeruginosa survival and biofilm formation. The crystal structure of PaDADH reveals the interaction of the glutamate 246 (E246) side chain with the substrate and at least three other active site residues, establishing a hydrogen bond network in the active site. Additionally, E246 likely ionizes to facilitate substrate binding during PaDADH catalysis. This study aimed to investigate how replacing the E246 residue with leucine affects PaDADH catalysis and its ability to react with O2 using steady-state kinetics coupled with pH profile studies. The data reveal a gain of O2 reactivity in the E246L variant, resulting in a reduced flavin semiquinone species and superoxide (O2•-) during substrate oxidation. The O2•- reacts with active site protons, resulting in an observed nonstoichiometric slope of 1.5 in the enzyme\'s log (kcat/Km) pH profile with D-arginine. Adding superoxide dismutase results in an observed correction of the slope to 1.0. This study demonstrates how O2•- can alter the slopes of limbs in the pH profiles of flavin-dependent enzymes and serves as a model for correcting nonstoichiometric slopes in elucidating reaction mechanisms of flavoproteins.
摘要：
酶是将生化反应速率提高几个数量级的有效催化剂。黄素蛋白是一类酶，其分类取决于它们在催化过程中使用可电离的活性位点残基与分子氧（O2）反应的能力。铜绿假单胞菌D-精氨酸脱氢酶(PaDADH)是一种黄素蛋白,其氧化D-精氨酸以用于铜绿假单胞菌存活和生物膜形成。PaDADH的晶体结构揭示了谷氨酸246（E246）侧链与底物和至少三个其他活性位点残基的相互作用，在活性位点建立氢键网络。此外,E246可能在PaDADH催化期间电离以促进底物结合。本研究旨在研究用亮氨酸代替E246残基如何影响PaDADH催化及其使用稳态动力学与pH曲线研究与O2反应的能力。数据显示E246L变体中O2反应性的增加，在底物氧化过程中导致黄素半醌物种和超氧化物（O2·-）减少。O2•-与活性位点质子反应,在D-精氨酸的酶的log(kcat/Km)pH曲线中观察到1.5的非化学计量斜率。添加超氧化物歧化酶导致观察到的斜率校正为1.0。这项研究证明了O2•-如何改变黄素依赖性酶的pH曲线中肢体的斜率，并作为校正非化学计量斜率的模型来阐明黄素蛋白的反应机理。
