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首页> 美国卫生研究院文献>Biochemical Journal >Pyruvate decarboxylase from Zymomonas mobilis. Structure and re-activation of apoenzyme by the cofactors thiamin diphosphate and magnesium ion.
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Pyruvate decarboxylase from Zymomonas mobilis. Structure and re-activation of apoenzyme by the cofactors thiamin diphosphate and magnesium ion.

机译：运动发酵单胞菌的丙酮酸脱羧酶。辅因子硫胺素二磷酸和镁离子的结构和再活化脱辅酶。

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摘要

To study the mechanism of re-activation of Zymomonas mobilis pyruvate decarboxylase apoenzyme by its cofactors thiamin diphosphate and Mg2+, cofactor-free enzyme was prepared by dialysis against 1 mM-dipicolinic acid at pH 8.2. This apoenzyme was then used in a series of experiments that included determination of: (a) the affinity towards one cofactor when the other was present at saturating concentrations; (b) cofactor-binding rates by measuring the quenching of tryptophan fluorescence on the apoenzyme; (c) the effect of replacement of cofactors with various analogues; (d) the stoichiometry of bound cofactors in holoenzyme; and (e) the molecular mass of apoenzyme by gel filtration. The results of these experiments form the basis for a proposed model for the re-activation of Z. mobilis pyruvate decarboxylase apoenzyme by its cofactors. In this model there exists two alterative but equivalent pathways for cofactor binding. In each pathway the first step is an independent reversible binding of either thiamin diphosphate (Kd 187 microM) or Mg2+ (Kd 1.31 mM) to free apoenzyme. When both cofactors are present, the second cofactor-binding step to form active holoenzyme is a slow quasi-irreversible step. This second binding step is a co-operative process for both thiamin diphosphate (Kd 0.353 microM) and Mg2+ (Kd 2.47 microM). Both the apo- and the holo-enzyme have a tetrameric subunit structure, with cofactors binding in a 1:1 ratio with each subunit.
机译：为了研究运动辅酶发酵单胞菌丙酮酸脱羧酶脱辅酶被辅酶硫胺素二磷酸和Mg2 +活化的机制，通过在pH 8.2下对1 mM-二吡啶甲酸进行渗析制备了无辅酶的酶。然后将该脱辅酶用于一系列实验，包括确定：（a）当另一种辅因子以饱和浓度存在时对一种辅因子的亲和力； （b）通过测量脱辅酶上色氨酸荧光的猝灭来辅助因子结合的速率； （c）用各种类似物代替辅因子的效果； （d）全酶中结合的辅因子的化学计量； （e）通过凝胶过滤的脱辅酶的分子量。这些实验的结果构成了拟议的运动发酵单胞菌丙酮酸脱羧酶脱辅酶辅因子再活化模型的基础。在该模型中，辅因子结合存在两种不同但等效的途径。在每种途径中，第一步是硫胺素二磷酸酯（Kd 187 microM）或Mg2 +（Kd 1.31 mM）与游离载脂酶的独立可逆结合。当两个辅因子都存在时，形成活性全酶的第二个辅因子结合步骤是一个缓慢的准不可逆步骤。这第二个结合步骤是硫胺素二磷酸盐（Kd 0.353 microM）和Mg2 +（Kd 2.47 microM）的合作过程。脱辅酶和全酶都具有四聚体亚基结构，辅因子与每个亚基的比例为1：1。

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