ALLOSTERIC INTERACTIONS IN ASPARTATE TRANSCARBAMYLASE .I. BINDING OF SPECIFIC LIGANDS TO NATIVE ENZYME AND ITS ISOLATED SUBUNITS
ALLOSTERIC INTERACTIONS IN ASPARTATE TRANSCARBAMYLASE .I. BINDING OF SPECIFIC LIGANDS TO NATIVE ENZYME AND ITS ISOLATED SUBUNITS
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DOI：
10.1021/bi00842a007
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发表时间：
1968-01-01
期刊：
影响因子：
2.9
通讯作者：
SCHACHMAN, HK
中科院分区：
文献类型：
--
作者：
CHANGEUX, JP;GERHART, JC;SCHACHMAN, HK
本文研究了大肠杆菌天冬氨酸转氨甲酰酶（ATCase）与配体的立体专一性相互作用。ATCase由2个催化亚基和4个调节亚基组成。平衡透析实验揭示了琥珀酸（底物天冬氨酸的类似物）的4个特异性结合位点和反馈抑制剂三磷酸胞苷（CTP）或其类似物5-溴三磷酸胞苷（BrCTP）/酶分子的4个特异性位点。基于这些结果和来自解离研究的证据，ATCase被视为异构体四聚体，其每个原聚体含有1个调节亚基（mol.重量2.7 x 104）和1/2的催化亚基（mol.重量5.0 x 104）。分离的调节亚基具有1个CTP结合位点，分离的催化亚基（mol.重量1 × 105）具有2个琥珀酸酯位点。尽管配体与分离的亚基的结合是正常的，但观察到这些相同的配体与天然酶的结合具有不寻常的作用。ATCase对琥珀酸盐结合表现出协同作用，如S形饱和曲线（希尔系数为1.6）所揭示的，以及拮抗作用，如琥珀酸盐部分减少CTP结合所揭示的。琥珀酸和CTP的结合发生在仅来自不同亚基的折叠多肽链的地形上不同的位点，因此协同和拮抗作用是间接的变构作用，其必须由蛋白质本身介导。与这些间接作用相反，ATCase还表现出直接作用，其中抑制剂（CTP）和激活剂三磷酸腺苷（ATP）似乎竞争调节亚基上的单个位点。
The regulatory enzyme aspartate transcarbamylase (ATCase) from Escherichia coli which is composed of 2 catalytic and 4 regulatory subunits was examined with regard to its stereospecific interactions with ligands. Equilibrium dialysis experiments reveal 4 specific binding sites for succinate (an analog of the substrate, aspartate) and 4 specific sites for the feedback inhibitor, cytidine triphosphate (CTP) or its analog, 5-bromocytidine triphosphate (BrCTP)/molecule of enzyme. On the basis of these results and evidence from dissociation studies, ATCase is viewed as an isologous tetramer, the protomers of which each contain 1 regulatory subunit (mol. wt. 2.7 x 104) and 1/2 of a catalytic subunit (mol. wt. 5.0 x 104). The isolated regulatory subunit possesses 1 binding site for CTP and the isolated catalytic subunit (mol. wt. 1 x 105) possesses 2 sites for succinate. Whereas the binding of ligands to the isolated subunits is normal, unusual effects are observed for the binding of these same ligands to the native enzyme. The ATCase exhibits cooperative effects for succinate binding as revealed by a sigmoidal saturation curve (with a Hill coefficient of 1.6) and antagonistic effects as revealed by a partial reduction of CTP binding by succinate. The binding of succinate and CTP occurs at topographically distinct sites derived exclusively from the folded polypeptide chains of the different subunits, and the cooperative and antagonistic effects are therefore indirect, allosteric effects which must be mediated by the protein itself. In contrast to these indirect effects, ATCase also exhibits a direct effect in which the inhibitor (CTP) and the activator adenosine triphosphate (ATP), appear to compete for a single site on the regulatory subunit.