Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida 32827.
Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico 88003.
J Biol Chem. 2020 May 8;295(19):6472-6481. doi: 10.1074/jbc.RA120.013198. Epub 2020 Mar 31.
The quinoprotein glycine oxidase from the marine bacterium (PlGoxA) uses a protein-derived cysteine tryptophylquinone (CTQ) cofactor to catalyze conversion of glycine to glyoxylate and ammonia. This homotetrameric enzyme exhibits strong cooperativity toward glycine binding. It is a good model for studying enzyme kinetics and cooperativity, specifically for being able to separate those aspects of protein function through directed mutagenesis. Variant proteins were generated with mutations in four active-site residues, Phe-316, His-583, Tyr-766, and His-767. Structures for glycine-soaked crystals were obtained for each. Different mutations had differential effects on and for catalysis, for substrate binding, and the Hill coefficients describing the steady-state kinetics or substrate binding. Phe-316 and Tyr-766 variants retained catalytic activity, albeit with altered kinetics and cooperativity. Substitutions of His-583 revealed that it is essential for glycine binding, and the structure of H583C PlGoxA had no active-site glycine present in glycine-soaked crystals. The structure of H767A PlGoxA revealed a previously undetected reaction intermediate, a carbinolamine product-reduced CTQ adduct, and exhibited only negligible activity. The results of these experiments, as well as those with the native enzyme and previous variants, enabled construction of a detailed mechanism for the reductive half-reaction of glycine oxidation. This proposed mechanism includes three discrete reaction intermediates that are covalently bound to CTQ during the reaction, two of which have now been structurally characterized by X-ray crystallography.
海洋细菌（PlGoxA）的醌蛋白甘氨酸氧化酶使用源自蛋白质的半胱氨酸色氨酸醌（CTQ）辅因子来催化甘氨酸转化为乙醛酸和氨。这种四聚体酶对甘氨酸结合表现出强烈的协同作用。它是研究酶动力学和协同作用的良好模型，特别是能够通过定向诱变来分离蛋白质功能的各个方面。在四个活性位点残基（苯丙氨酸 316、组氨酸 583、酪氨酸 766 和组氨酸 767）上生成了变体蛋白。获得了每种变体蛋白的甘氨酸浸泡晶体的结构。不同的突变对催化的 和 、底物结合的 以及描述稳态动力学或底物结合的希尔系数产生了不同的影响。苯丙氨酸 316 和酪氨酸 766 变体保留了催化活性，尽管动力学和协同性发生了改变。组氨酸 583 的取代表明它对甘氨酸结合至关重要，并且在甘氨酸浸泡晶体中 H583C PlGoxA 的结构中没有存在活性部位甘氨酸。H767A PlGoxA 的结构揭示了一个以前未检测到的反应中间体，即碳醇胺产物还原的 CTQ 加合物，并且仅表现出可忽略不计的活性。这些实验的结果，以及天然酶和以前变体的结果，使构建甘氨酸氧化还原半反应的详细机制成为可能。该提议的机制包括在反应过程中与 CTQ 共价结合的三个离散反应中间体，其中两个现已通过 X 射线晶体学结构表征。
