Mechanism of allosteric transition of bacterial L-lactate dehydrogenase.
Mechanism of allosteric transition of bacterial L-lactate dehydrogenase.
复制标题
细菌L-乳酸脱氢酶变构转变机制。
作者：
T. Ohta;K. Yokota;T. Minowa;S. Iwata
通过亚基杂交技术和X射线晶体学研究了长双歧杆菌aM101-2的L-乳酸脱氢酶（L-乳酸：NAD+氧化还原酶，EC 1.1.1.27，LDH）的变构行为。 LDH 活性的同向变构反应与其底物丙酮酸的浓度有关。通过添加果糖 1,6-二磷酸 (FBP) 诱导酶的异向变构激活。通过定点诱变制备了两种突变酶，它们要么改变了对 FBP 的敏感性，要么改变了从丙酮酸到草酰乙酸的底物特异性。野生型和突变型LDH之间的杂种LDH是通过使用带有野生型或突变型LDH基因的两个质粒对大肠杆菌进行双重转化，通过体内亚基杂交制备的。仅将一种脱敏突变亚基引入LDH就将野生酶的特性改变为突变LDH的特性。对由具有不同底物特异性的亚基组成的杂合酶的动力学研究表明，亚基之间存在很强的协同相互作用。这些结果有力地支持了 LDH 的变构变化符合 Monod、Wyman 和 Changeux 提出的协同对称模型的观点。我们以 1.9 A 分辨率分析了对底物具有低亲和力的 LDH 的晶体结构。通过与其他LDH结构的比较，我们得出结论：LDH的构象转变主要是由亚基的协同旋转引起的。
The allosteric behaviour of L-lactate dehydrogenase (L-lactate:NAD+oxidoreductase, EC 1.1.1.27, LDH) from Bifidobacterium longum aM101-2 was studied by means of the subunit hybridization technique as well as X-ray crystallography. Homotropic allosteric response of the LDH activity was found against the concentration of its substrate, pyruvate. Heterotropic allosteric activation of the enzyme was induced by the addition of fructose 1,6-bisphosphate (FBP). Two mutant enzymes, which had either altered sensitivity to FBP or altered substrate specificity from pyruvate to oxaloacetate, were made by site-directed mutagenesis. Hybrid LDHs, between the wild and the mutant LDHs, were made by in vivo subunit hybridization using double transformation of Escherichia coli with two plasmids carrying either the gene of wild or mutant LDH. Introduction of only one desensitized mutant subunit to the LDH changed the characteristic of the wild enzyme to that of the mutant LDH. Kinetic studies on hybrid enzymes consisting of subunits having different substrate specificity indicated that there was a strong cooperative interaction among subunits. These results strongly support the idea that the allosteric change of the LDH fits the concerted-symmetry model proposed by Monod, Wyman, and Changeux. We analysed the crystallographic structure of the LDH having low affinity to substrate at 1.9 A resolution. By the comparison of the structure with that of other LDHs, we concluded that the conformational transition of the LDH was mainly caused by concerted rotations of subunits.
影响因子：
2.9
作者：
GOUAUX, JE;LIPSCOMB, WN
通讯作者：
LIPSCOMB, WN
影响因子：
5.6
作者：
GRAU, UM;TROMMER, WE;ROSSMANN, MG
通讯作者：
ROSSMANN, MG