Tauc P, Vachette P, Middleton S A, Kantrowitz E R
Institut d'Enzymologie, C.N.R.S., Gif Sur Yvette, France.
J Mol Biol. 1990 Jul 5;214(1):327-35. doi: 10.1016/0022-2836(90)90164-H.
Low-angle X-ray scattering in solution has been used to probe the quaternary structure of a mutant version of Escherichia coli aspartate transcarbamylase in which Glu239 of the catalytic chain was replaced by glutamine by site-directed mutagenesis. X-ray crystallographic studies of the wild-type enzyme have shown that one set of intersubunit interactions involving Glu239 are lost, and are replaced by another set of intrachain interactions when the enzyme undergoes the allosteric transition from the T to the R state. Functional analysis of the mutant enzyme with glutamine in place of Glu239 indicates that homotropic co-operativity is lost without altering the maximal specific activity. The radius of gyration of the unligated mutant enzyme is larger than the unligated wild-type, indicating an alteration in quaternary structure of the mutant. However, the radius of gyration of the mutant enzyme in the presence of N-(phosphonoacetyl)-L-aspartate (PALA) is identical with the value for the wild-type enzyme in the presence of PALA. X-ray scattering at larger angles indicates that the mutant enzyme is in a new structural state different from the wild-type T and R structures. The scattering pattern in the presence of saturating concentrations of PALA is identical with that of the wild-type R structure. Saturating concentrations of carbamyl phosphate alone are sufficient to convert most of the mutant enzyme to the R structure, in the absence of aspartate. CTP shifts the scattering pattern of the mutant enzyme in the presence of saturating carbamyl phosphate towards the scattering curve of the unligated enzyme, but CTP has no effect on the scattering curve in the absence of carbamyl phosphate or in the presence of subsaturating PALA. However, in the presence of subsaturating PALA, ATP causes a strong shift towards the R structure. Neither ATP nor CTP has any effect on the activity of the mutant enzyme. These data suggest that the replacement of Glu239 by glutamine results in a new quaternary structure. These data also explain, on a structural basis, why co-operativity is lost in this mutant enzyme.
溶液中的小角X射线散射已被用于探测大肠杆菌天冬氨酸转氨甲酰酶突变体的四级结构，该突变体通过定点诱变将催化链中的Glu239替换为谷氨酰胺。野生型酶的X射线晶体学研究表明，当酶从T态向R态进行别构转变时，涉及Glu239的一组亚基间相互作用丧失，并被另一组链内相互作用所取代。对用谷氨酰胺取代Glu239的突变酶进行功能分析表明，同源协同性丧失，但最大比活性未改变。未结合配体的突变酶的回转半径大于未结合配体的野生型酶，表明突变体的四级结构发生了改变。然而，在存在N-（膦酰基乙酰基）-L-天冬氨酸（PALA）的情况下，突变酶的回转半径与存在PALA的野生型酶的值相同。较大角度的X射线散射表明，突变酶处于不同于野生型T和R结构的新结构状态。在存在饱和浓度PALA的情况下的散射模式与野生型R结构的散射模式相同。仅饱和浓度的氨甲酰磷酸就足以在没有天冬氨酸的情况下将大多数突变酶转化为R结构。CTP使存在饱和氨甲酰磷酸的突变酶的散射模式向未结合配体的酶的散射曲线移动，但CTP在没有氨甲酰磷酸或存在亚饱和PALA的情况下对散射曲线没有影响。然而，在存在亚饱和PALA的情况下，ATP会导致向R结构的强烈移动。ATP和CTP对突变酶的活性均无任何影响。这些数据表明，用谷氨酰胺取代Glu239会导致新的四级结构。这些数据还从结构基础上解释了为什么这种突变酶会丧失协同性。
