Jacobsverg L B, Kantrowitz E R, Lipscomb W N
J Biol Chem. 1975 Dec 25;250(24):9238-49.
The interaction of the dye tetraiodofluorescein with native aspartate transcarbamylase and its isolated subunits has been investigated by both binding and activity measurements at 4 and 23 degrees. At room temperature low concentrations of tetraiodofluorescein activate the native enzyme, but high concentrations inhibit the enzyme's activity. At the low temperature the native enzyme is inhibited by all concentrations of dye. Isolated catalytic subunit is very effectively inhibited at both temperatures. For the native enzyme these results are explained by 18 tetraiodofluorescein sites of approximately equal affinity (K = 7.3 X 10(-5) M) on each enzyme hexamer: one class of six sites at the nucleoside triphosphate effector binding sites is responsible for the activation, a second class which competes with the substrate carbamylphosphate causes the inhibition, and a third class does not interact with either the effectors or the substrates. Measurements of tetraiodofluorescein binding to isolated regulatory, catalytic, and p-hydroxymercuribenzoate-inactivated catalytic subunits support the above assignments. This scheme of tetraiodofluorescein binding sites successfully predicts the changes in the tetraiodofluorescein-aspartate transcarbamylase difference spectrum induced by the addition of various ligands. The activity changes induced by the dye are explained if the binding of a single tetraiodofluorescein molecule to one of the six regulatory sites activates all six of the catalytic sites, while while a dye molecule binding to any one of the catalytic sites inactivates only that catalytic site.
已通过在4摄氏度和23摄氏度下的结合及活性测量，研究了染料四碘荧光素与天然天冬氨酸转氨甲酰酶及其分离亚基的相互作用。在室温下，低浓度的四碘荧光素激活天然酶，但高浓度则抑制该酶的活性。在低温下，所有浓度的染料都会抑制天然酶。分离的催化亚基在这两个温度下都受到非常有效的抑制。对于天然酶，这些结果可通过每个酶六聚体上18个亲和力大致相等的四碘荧光素位点（K = 7.3×10⁻⁵ M）来解释：一类六个位于核苷三磷酸效应物结合位点的位点负责激活，与底物氨甲酰磷酸竞争的第二类位点导致抑制，第三类位点与效应物或底物均不相互作用。对四碘荧光素与分离的调节亚基、催化亚基以及对羟基汞苯甲酸失活的催化亚基的结合测量支持上述归属。这种四碘荧光素结合位点的方案成功预测了添加各种配体引起的四碘荧光素 - 天冬氨酸转氨甲酰酶差光谱的变化。如果单个四碘荧光素分子与六个调节位点之一的结合激活所有六个催化位点，而染料分子与任何一个催化位点的结合仅使该催化位点失活，那么就可以解释染料引起的活性变化。
