Characterization of the covalent enzyme intermediates formed during pyruvate phosphate dikinase catalysis.
Characterization of the covalent enzyme intermediates formed during pyruvate phosphate dikinase catalysis.
复制标题
丙酮酸磷酸二激酶催化过程中形成的共价酶中间体的表征。
DOI：
10.1021/bi00058a014
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Dunaway-Mariano,D
中科院分区：
文献类型：
--
作者：
Thrall,SH;Mehl,AF;Carroll,LJ;Dunaway-Mariano,D
摘要：利用瞬态动力学技术研究了焦磷酸酶（E-PP）和磷酸酶（EP）在共生梭菌丙酮酸磷酸二激酶催化5′-三磷酸腺苷（ATP）、正磷酸（P′）和丙酮酸与S′-单磷酸腺苷（AMP）、无机焦磷酸（PR）和磷酸烯醇丙酮酸（PEP）相互转化中的中间作用。用[-32]或[14C] ATP和PPDK分别在P存在和不存在的情况下进行单次翻转实验，检测焦磷酸化酶和AMP的形成。发现E-PP·AMP复合物的形成之后是P的结合和EP-AMP* PP复合物的形成。发现单次转化过程中积累的焦磷酶水平依赖于所使用的二价金属辅助因子（Mn2+> Co2+> Mg2+）。在PP +丙酮酸存在和不存在的情况下，进行了[32P] PEP和PPDK的单次翻转实验，以检测在相反的反应方向上形成磷酸酶，形成atp。由E-PEP复合物反应形成的磷酸化酶在AMP和不含PP的酶存在下以超过稳态周转率的速率转化。测定了丙酮磷酸二激酶的反应顺序为p′
Revised Manuscript Received November 23, 1992 abstract: The intermediacy of a pyrophosphorylenzyme (E-PP) and phosphorylenzyme (EP) in the Clostridium symbiosum pyruvate phosphate dikinase catalyzed interconversion of adenosine 5'-triphosphate (ATP), orthophosphate (P¡), and pyruvate with adenosine S'-monophosphate (AMP), inorganic pyrophosphate (PR), and phosphoenolpyruvate (PEP) was examined using transientkinetic techniques. Single-turnover experiments with [-32] or [14C] ATP and PPDK were carried out in the presence and absence of P¡ to test for pyrophosphorylenzyme and AMP formation, respectively. Formation of the E-PP· AMP complex was found to be followed by P¡ binding and the formation of the EP-AMP* PP¡ complex. The level of pyrophosphorylenzyme accumulated during a single turnover was found to be dependent on the divalent metal cofactor used (Mn2+> Co2+> Mg2+). Single-turnover experiments with [32P] PEP and PPDK were carried out in thepresence and absence of PP¡ and pyruvate to test for phosphorylenzyme formation in the reverse, ATP-forming direction of the reaction. Phosphorylenzyme formed from the reaction of the E-PEP complex was converted in the presence of AMP and PP¡ to free enzyme at a rate exceeding the steady-state turnover rate. The reaction sequence for pyrvate phosphate dikinase was determined to be p¡