HOMOTROPIC EFFECTS IN ASPARTATE TRANSCARBAMOYLASE - WHAT HAPPENS WHEN THE ENZYME BINDS A SINGLE MOLECULE OF THE BISUBSTRATE ANALOG N-PHOSPHONACETYL-L-ASPARTATE
HOMOTROPIC EFFECTS IN ASPARTATE TRANSCARBAMOYLASE - WHAT HAPPENS WHEN THE ENZYME BINDS A SINGLE MOLECULE OF THE BISUBSTRATE ANALOG N-PHOSPHONACETYL-L-ASPARTATE
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DOI：
10.1016/0022-2836(85)90267-0
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发表时间：
1985-01-01
影响因子：
5.6
通讯作者：
SCHACHMAN, HK
中科院分区：
文献类型：
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作者：
FOOTE, J;SCHACHMAN, HK
天冬氨酸转氨甲酰酶从大肠杆菌的活性位点进行滴定，通过测量减少酶催化的砷解N-氨甲酰基-L-天冬氨酸引起的除了紧密结合的抑制剂。N-膦酰基-L-天冬氨酸。因为这种酶对于这种非生理反应来说催化剂很差。试验需要高浓度（超过可逆结合抑制剂解离常数的1000倍），因此，几乎所有的双底物类似物都被结合。从滴定的终点，计算出5.7个活性位点，与基于酶结构的6个数目非常一致。只有当抑制剂与酶的摩尔比超过5时才观察到简单的抑制：在这些条件下，如早期的理化研究所示，酶的R-构象状态是唯一或主要的物种。在低比率的抑制剂酶，抑制剂的添加引起的活性增加，这是由于酶从低活性的T-状态的转换到更活跃的R-状态。在低摩尔比（0.01，即1个抑制剂600个活性位点）下作为抑制剂浓度的函数的活性的线性增加与在高摩尔比下的活性损失的比较提供了作为一种双底物类似物与酶分子结合的结果的从T-状态转化为R-状态的活性位点的平均数的直接值。这个数字是四与毫克。ATP或氨基甲酰磷酸存在下的酶和4.7在Mg. PPi，值接近或相同的理论最大值，4.7的协同过渡与所有的活性位点的分子从T-到R-状态的变化后，形成一个二元复合物的六聚体酶与一个单一的抑制剂。在不存在效应物的情况下用酶或用Mg. CTP存在时，滴定显示，两个和一个位点的平均值分别为4.7，可能在配体结合后改变构象。这些结果被解释为一个亚群的T-和R-状态的酶复合物含有一个绑定的抑制剂分子之间的平衡的表现。在不存在外源配体的情况下，R-状态物质将代表天冬氨酸转氨甲酰酶群体的40%。Mg. ATP和Mg.当酶与一个双底物类似物分子结合时，CTP分别增加和减少R-态活性位点的表观产率，这与以前的研究结果（ATP优先结合R-态，CTP优先结合T-态）雅阁。
The active sites of aspartate transcarbamoylase from Escherichia coli were titrated by measuring the decrease in the enzyme-catalyzed arsenolysis of N-carbamoyl-L-aspartate caused by the addition of the tight-binding inhibitor. N-phosphonacetyl-L-aspartate. Because the enzyme is a poor catalyst for this non-physiological reaction. High concentrations are required for the assays (more than 1000-fold the dissociation constant of the reversibly bound inhibitor) and, therefore, virtually all of the bisubstrate analog is bound. From the endpoint of the titration, 5.7 active sites were calculated, in excellent agreement with the number, six, based on the structure of the enzyme. Simple inhibition was observed only when the molar ratio of inhibitor to enzyme exceeded five: under these conditions, as shown in earlier physicl chemical studies, the R-conformational state of the enzyme is the sole or predominant species. At low ratios of inhibitor to enzyme, the addition of inhibitor caused an increase in activity which is attributable to the conversion of the enzyme from the low-activity T-state to the much more active R-state. Comparison of the linear increase in activity as a function of inhibitor concentration at the low molar ratio (0.01, i.e. 1 inhibitor 600 active sites) with the activity lost at the high ratio provided a direct value for the mean number of active sites converted from the T-state to the R-state as a result of the binding of one bisubstrate analog to an enzyme molecule. This number was four with Mg .cntdot. ATP or carbamoyl phosphate present and 4.7 for the enzyme in the presence of Mg .cntdot. PPi, values approaching or identical to the theoretical maximum, 4.7 for a concerted transition with all of the active sites of the molecule changing from the T- to R-state upon the formation of a binary complex of hexameric enzyme with a single inhibitor. With the enzyme in the absence of effectors or with Mg .cntdot. CTP present, the titrations showed that an average of two and one sites, respectively, of 4.7 possible, changed conformation upon ligand binding. These results were interpreted as a manifestation of an equilibrium between a sub-population of T- and R-state enzyme complexes containing one bound inhibitor molecule. The R-state species would represent 40% of the population for aspartate transcarbamoylase in the absence of extraneous ligands. The effects of Mg .cntdot. ATP and Mg .cntdot. CTP in increasing and decreasing, respectively, the apparent yield of R-state active sites when the enyzme binds one bisubstrate analog molecule are in accord with previous findings of the preferential binding of ATP to the R-state and CTP to the T-state.