von Döbeln U
Biochemistry. 1977 Oct 4;16(20):4368-71. doi: 10.1021/bi00639a006.
Ribonucleotide reductase is responsible for the production of deoxyribonucleotides by catalyzing the reduction of ribonucleoside diphosphates. The enzyme is allosterically regulated in a complex way by the nucleoside triphosphates, ATP, dTTP, dGTP, dCTP, and dATP. Ribonucleotide reductase consists of two nonidentical subunits, proteins B1 and B2. Both substrates and allosteric effectors bind exclusively to B1. Binding of protein B1 to dTTP or dATP covalently coupled to Sepharose and elution with concentration gradients of the different nucleoside triphosphate effectors gave information about (1) the arrangement of the effector binding sites on protein B1 and (2) the affinity of the effectors for these sites. Protein B1 thus has two classes of effector binding sites. One class binds all effectors, as demonstrated by elution of the protein from dTTP-Sepharose with dATP, dGTP, ATP, or dCTP. The second class binds only dATP or ATP, since dATP and ATP were the only nucleotides which eluted protein B1 from dATP-Sepharose. These results confirm earlier data obtained by dialysis binding experiments. The eluting concentrations obtained for the different nucleoside triphosphates in experiments with dTTP-Sepharose could be used to calculate unknown dissociation constants for protein B1 -effector binary complexes. This was possible, since a plot of the eluting concentrations vs. known dissociation constants was linear.
核糖核苷酸还原酶负责通过催化核糖核苷二磷酸的还原反应来生成脱氧核糖核苷酸。该酶受到核苷三磷酸、ATP、dTTP、dGTP、dCTP和dATP以复杂的变构方式调节。核糖核苷酸还原酶由两个不同的亚基，即蛋白质B1和B2组成。底物和变构效应物都仅与B1结合。将与琼脂糖共价偶联的蛋白质B1与dTTP或dATP结合，并使用不同核苷三磷酸效应物的浓度梯度进行洗脱，可得到有关（1）蛋白质B1上效应物结合位点的排列以及（2）效应物与这些位点的亲和力的信息。因此，蛋白质B1有两类效应物结合位点。一类结合所有效应物，这可通过用dATP、dGTP、ATP或dCTP从dTTP - 琼脂糖上洗脱蛋白质来证明。第二类仅结合dATP或ATP，因为dATP和ATP是仅有的能从dATP - 琼脂糖上洗脱蛋白质B1的核苷酸。这些结果证实了早期通过透析结合实验获得的数据。在dTTP - 琼脂糖实验中获得的不同核苷三磷酸的洗脱浓度可用于计算蛋白质B1 - 效应物二元复合物的未知解离常数。这是可行的，因为洗脱浓度与已知解离常数的关系图是线性的。
