Rapid reaction kinetics of proline dehydrogenase in the multifunctional proline utilization A protein.
Rapid reaction kinetics of proline dehydrogenase in the multifunctional proline utilization A protein.
复制标题
多功能脯氨酸利用A蛋白质中脯氨酸脱氢酶的快速反应动力学。
DOI：
10.1021/bi201603f
复制
发表时间：
2012-01-10
期刊：
影响因子：
2.9
通讯作者：
Becker, Donald F.
中科院分区：
文献类型：
--
作者：
Moxley, Michael A.;Becker, Donald F.
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（P5 C）脱氢酶结构域在两个反应步骤中催化脯氨酸氧化成谷氨酸。在这里，在PutA的PRODH的动力学机制研究停流动力学，以确定微观速率常数的脯氨酸：泛醌氧化还原酶机制。停流数据脯氨酸还原的黄素辅因子（还原半反应）和辅酶Q1氧化还原黄素（氧化半反应）是最适合的双指数，从最大可观察到的速率常数和表观平衡解离常数进行了测定。在还原或氧化反应中未观察到黄素半醌。微观速率常数的步骤中的还原和氧化半反应，获得了全局拟合的停流数据的模拟机制，包括一个化学步骤，然后由异构化事件。黄素辅因子的脯氨酸还原的微观速率常数为27.5 s-1，随后的异构化步骤为2.2 s-1。提出异构化步骤以报道先前鉴定的黄素依赖性构象变化（Zhang，W.等人（2007）Biochemistry 46，483-491），其对于PutA功能转换是重要的，但与体外机制在动力学上不相关。使用辅酶Q1，一种泛醌的可溶性类似物，获得了黄素氧化的速率常数为5.4 s−1，从而表明该氧化步骤是催化周转期间kcat的限速步骤。从微观速率常数计算的稳态动力学常数与实验kcat和kcat/Km参数一致。
The multifunctional proline utilization A (PutA) flavoenzyme from Escherichia coli catalyzes the oxidation of proline to glutamate in two reaction steps using separate proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate (P5C) dehydrogenase domains. Here, the kinetic mechanism of PRODH in PutA is studied by stopped-flow kinetics to determine microscopic rate constants for the proline:ubiquinone oxidoreductase mechanism. Stopped-flow data for proline reduction of the flavin cofactor (reductive half-reaction) and oxidation of reduced flavin by CoQ1 (oxidative half-reaction) were best-fit by a double exponential from which maximum observable rate constants and apparent equilibrium dissociation constants were determined. Flavin semiquinone was not observed in the reductive or oxidative reactions. Microscopic rate constants for steps in the reductive and oxidative half-reactions were obtained by globally fitting the stopped-flow data to a simulated mechanism that includes a chemical step followed by an isomerization event. A microscopic rate constant of 27.5 s−1 was determined for proline reduction of the flavin cofactor followed by an isomerization step of 2.2 s−1. The isomerization step is proposed to report on a previously identified flavin-dependent conformational change (Zhang, W. et al. (2007) Biochemistry 46, 483–491) that is important for PutA functional switching but is not kinetically relevant to the in vitro mechanism. Using CoQ1, a soluble analog of ubiquinone, a rate constant of 5.4 s−1 was obtained for the oxidation of flavin, thus indicating that this oxidative step is rate-limiting for kcat during catalytic turnover. Steady-state kinetic constants calculated from the microscopic rate constants agree with the experimental kcat and kcat/Km parameters.
登录
查看更多内容
影响因子：
3.9
作者：
Moxley MA;Tanner JJ;Becker DF
通讯作者：
Becker DF
影响因子：
8
作者：
Liu, Y.;Borchert, G. L.;Phang, J. M.
通讯作者：
Phang, J. M.
影响因子：
2.9
作者：
MEZL, VA;KNOX, WE
通讯作者：
KNOX, WE
影响因子：
3.4
作者：
Surber, MW;Maloy, S
通讯作者：
Maloy, S
DOI：
10.1093/jn/138.10.2008s
发表时间：
2008-10
期刊：
The Journal of nutrition
影响因子：
--
作者：
Phang JM;Pandhare J;Liu Y
通讯作者：
Liu Y