The quinoprotein glycine oxidase from the marine bacterium Pseudoalteromonas luteoviolacea (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 k cat and K 0.5 for catalysis, K 0.5 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.

中文翻译：

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活性位点残基在喹蛋白甘氨酸氧化酶的催化，底物结合，协同作用和反应机理中的作用。

来自海洋细菌Pseudoalteromonas luteoviolacea（PlGoxA）的喹蛋白甘氨酸氧化酶使用蛋白质衍生的半胱氨酸色氨酸醌（CTQ）辅助因子催化甘氨酸向乙醛酸和氨的转化。该同四聚体酶对甘氨酸结合表现出很强的协同性。这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分离蛋白质功能的那些方面。产生了具有四个活性位点残基突变的变异蛋白，即Phe-316，His-583，Tyr-766和His-767。分别获得了甘氨酸浸泡的晶体的结构。不同的突变对k cat和K 0.5的催化作用，对K 0.5的底物结合有不同的影响，希尔系数描述了稳态动力学或底物结合。Phe-316和Tyr-766变体保留了催化活性，尽管动力学和协同性发生了变化。His-583的取代表明它对于甘氨酸结合至关重要，并且H583C PlGoxA的结构在甘氨酸浸泡的晶体中不存在活性位点甘氨酸。H767A PlGoxA的结构显示了以前未发现的反应中间体，即甲醇胺产物还原的CTQ加合物，并且仅表现出微不足道的活性。这些实验以及天然酶和以前的变体的实验结果使得能够构建用于甘氨酸氧化还原半反应的详细机理。该提出的机理包括在反应过程中共价键合到CTQ的三个离散反应中间体，其中两个现在已经通过X射线晶体学进行了结构表征。