Steady-state kinetic mechanism of the proline:ubiquinone oxidoreductase activity of proline utilization A (PutA) from Escherichia coli.
Steady-state kinetic mechanism of the proline:ubiquinone oxidoreductase activity of proline utilization A (PutA) from Escherichia coli.
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DOI：
10.1016/j.abb.2011.10.011
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发表时间：
2011-12-15
影响因子：
3.9
通讯作者：
Becker DF
中科院分区：
文献类型：
--
作者：
Moxley MA;Tanner JJ;Becker DF
来自大肠杆菌的多功能脯氨酸利用 A (PutA) 黄素酶使用单独的脯氨酸脱氢酶 (PRODH) 和 Δ1-吡咯啉-5-羧酸酯 (P5C) 脱氢酶结构域在两个催化步骤中将脯氨酸氧化为谷氨酸。在第一个反应中，脯氨酸的氧化与 PRODH 结构域对泛醌 (CoQ) 的还原偶联，该结构域具有在细菌和真核 PRODH 酶中保守的 β8α8 桶结构。使用 CoQ 类似物通过稳态动力学检查苯醌部分的结构要求。 PutA 显示了所有测试类似物的活性；使用 CoQ2 获得了最高的 kcat/Km。使用多种稳态方法研究了 PRODH 反应的动力学机制。使用脯氨酸和 CoQ1 测量的初始速度模式，结合死端和产物抑制研究，表明 PutA 存在两个位点乒乓机制。 PutA 的动力学参数不受溶剂粘度的强烈影响，表明扩散步骤不会显着限制总体反应速率。总之，此处报告的动力学数据以及 PRODH 结构域的晶体结构数据分析表明，PutA 的脯氨酸：泛醌氧化还原酶反应通过脯氨酸和泛醌在两个不同位点结合的快速平衡乒乓机制发生。
The multifunctional proline utilization A (PutA) flavoenzyme from Escherichia coli performs the oxidation of proline to glutamate in two catalytic steps using separate proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate (P5C) dehydrogenase domains. In the first reaction, the oxidation of proline is coupled to the reduction of ubiquinone (CoQ) by the PRODH domain, which has a β8α8-barrel structure that is conserved in bacterial and eukaryotic PRODH enzymes. The structural requirements of the benzoquinone moiety were examined by steady-state kinetics using CoQ analogs. PutA displayed activity with all the analogs tested; the highest kcat/Km was obtained with CoQ2. The kinetic mechanism of the PRODH reaction was investigated use a variety of steady-state approaches. Initial velocity patterns measured using proline and CoQ1, combined with dead-end and product inhibition studies, suggested a two-site ping-pong mechanism for PutA. The kinetic parameters for PutA were not strongly influenced by solvent viscosity suggesting that diffusive steps do not significantly limit the overall reaction rate. In summary, the kinetic data reported here, along with analysis of the crystal structure data for the PRODH domain, suggest that the proline:ubiquinone oxidoreductase reaction of PutA occurs via a rapid equilibrium ping-pong mechanism with proline and ubiquinone binding at two distinct sites.
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影响因子：
3.9
作者：
HAGEDORN, CH;PHANG, JM
通讯作者：
PHANG, JM
影响因子：
4.8
作者：
Gu, D;Zhou, YZ;Becker, DF
通讯作者：
Becker, DF
DOI：
10.1111/j.1432-1033.1980.tb04476.x
发表时间：
1980-01-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
COUGHLAN, MP;RAJAGOPALAN, KV
通讯作者：
RAJAGOPALAN, KV
影响因子：
2.9
作者：
HINES, V;JOHNSTON, M
通讯作者：
JOHNSTON, M
影响因子：
2.2
作者：
HARGROVE, JW
通讯作者：
HARGROVE, JW