Stebbins J W, Kantrowitz E R
Department of Chemistry, Boston College, Massachusetts 02167.
J Biol Chem. 1989 Sep 5;264(25):14860-4.
Recent x-ray crystallographic studies of aspartate transcarbamoylase bound with CTP have detected molecular asymmetry in the interface between the catalytic and regulatory subunits (Kim, K. H., Pan, Z., Honzatko, R. B., Ke, H.-M., and Lipscomb, W. N. (1987) J. Mol. Biol. 196, 863-875). In three of the six interfaces, a salt link occurs between Arg130 of the regulatory chain and Glu204 of the catalytic chain; however, these same residues are 15 A apart in the other three interfaces. In order to determine if this is important for the function of the enzyme, two mutant versions of aspartate transcarbamoylase were created by site-specific mutagenesis. Glu204 of the catalytic chain was converted to a glutamine (Glu204c----Gln) and Arg130 of the regulatory chain was converted to a glycine (Arg130r----Gly). The thermal stability of the Arg130r----Gly enzyme is dramatically reduced, whereas the thermal stability of the Glu204c----Gln enzyme is unaltered compared to the wild-type enzyme. The maximal velocity of both mutant enzymes is identical with that of the wild-type enzyme, however both mutant enzymes have altered substrate affinity and regulatory properties. Based on these studies, the link between Glu204 of the catalytic chain and Arg130 of the regulatory chain is important for the heterotropic properties of the enzyme. Furthermore, the interface between the domain of the regulatory chain which binds zinc and the domain of the catalytic chain which binds aspartate may be more important for CTP inhibition than ATP activation. These data also suggest that heterotropic cooperativity is very sensitive to alterations in the catalytic-regulatory interface. However, no clear relationship has been observed between the structural asymmetry and the function of the enzyme.
最近对与CTP结合的天冬氨酸转氨甲酰酶进行的X射线晶体学研究，在催化亚基和调节亚基之间的界面检测到分子不对称性（Kim, K. H., Pan, Z., Honzatko, R. B., Ke, H.-M., and Lipscomb, W. N. (1987) J. Mol. Biol. 196, 863 - 875）。在六个界面中的三个界面，调节链的Arg130与催化链的Glu204之间形成了盐桥；然而，在其他三个界面中，相同的残基相距15埃。为了确定这对酶的功能是否重要，通过定点诱变创建了两个天冬氨酸转氨甲酰酶的突变体版本。催化链的Glu204被转化为谷氨酰胺（Glu204c----Gln），调节链的Arg130被转化为甘氨酸（Arg130r----Gly）。与野生型酶相比，Arg130r----Gly酶的热稳定性显著降低，而Glu204c----Gln酶的热稳定性未改变。两种突变酶的最大速度与野生型酶相同，然而两种突变酶都改变了底物亲和力和调节特性。基于这些研究，催化链的Glu204与调节链的Arg130之间的联系对酶的异促性质很重要。此外，结合锌的调节链结构域与结合天冬氨酸的催化链结构域之间的界面，对于CTP抑制可能比ATP激活更重要。这些数据还表明，异促协同性对催化 - 调节界面的改变非常敏感。然而，在结构不对称性与酶的功能之间未观察到明确的关系。
