Buttlaire D H, Reed G H, Himes R H
J Biol Chem. 1975 Jan 10;250(1):254-60.
Binding of Mn(pi)-nucleotide complexes to the enzyme formyltertrahydrofolate synthetase (EC 6.3.4.3) from Clostridium cylindrosporum has been examined in the presence and absence of other substrates by solvent proton relaxation mearurements. MnADP and MnATP form ternary complexes with the enzyme with highly enhanced proton relaxation rates for water. The enhancement parameters, epsilont, for the MnADP and MnATP ternary complexes are 19.8 and 12.5, respectively at 24.3 MHZ and 25 degrees. Titration curves with constant total concentrations of enzyme and Mn(pi) with variable nucleotide concentration are similar to those observed in similar titrations with the endp and MnATP are 175 muM and 64 muM, respectively at 25 degrees. Addition of tetrahydrofolate to solutions of the MnADP OR MnATP ternary complexes lowers the observed relaxation enhancement markedly. An analysis of titration curves with constant total concentrations of enzyme, Mn(pi), and nucleotide with variable tetrahydrofolate concentration gives the dissociation constant for tetrahydrofolate from the respective quaternary complexes. The affinity of the enzyme for tetrahydrofolate is increased 6-fold when MnADP is present at the active site whereas a 3-fold increase is observed with MnATP present. Furthermore, there is a 20-fold increase in the enzyme's affinity for tetrahydrofolate when both MnADP and the third substrate, formate, are present. The observed relaxation rate of water for solutions of the complex, enzyme-MnADP-tetrahydrofolate-formate, is deenhanced with respect to the rate observed for the simple aquo-Mn(pi) solution. Addition of nitrate to solutions of the above complex increases the affinity of the enzyme for tetrahydrofolate and MnADP by an additional factor of 5 and lowers the relaxation rate further to a value which approaches that for solutions of the enzyme and substrates which lack the paramagnetic cation.
通过溶剂质子弛豫测量，研究了在有或没有其他底物存在的情况下，锰（Ⅱ）-核苷酸复合物与来自柱状芽孢梭菌的甲酰四氢叶酸合成酶（EC 6.3.4.3）的结合情况。锰ADP和锰ATP与该酶形成三元复合物，使水的质子弛豫速率大幅提高。在24.3兆赫兹和25摄氏度下，锰ADP和锰ATP三元复合物的增强参数ε分别为19.8和12.5。在酶和锰（Ⅱ）总浓度恒定、核苷酸浓度可变的情况下进行滴定曲线测定，其结果与用终产物和锰ATP进行类似滴定所观察到的曲线相似，在25摄氏度下，解离常数分别为175微摩尔和64微摩尔。向锰ADP或锰ATP三元复合物溶液中添加四氢叶酸会显著降低观察到的弛豫增强。在酶、锰（Ⅱ）和核苷酸总浓度恒定、四氢叶酸浓度可变的情况下进行滴定曲线分析，得出了四氢叶酸从相应四元复合物中的解离常数。当活性位点存在锰ADP时，酶对四氢叶酸的亲和力增加了6倍，而当存在锰ATP时，观察到亲和力增加了3倍。此外，当锰ADP和第三种底物甲酸盐同时存在时，酶对四氢叶酸的亲和力增加了20倍。与简单的水合锰（Ⅱ）溶液相比，复合物（酶-锰ADP-四氢叶酸-甲酸盐）溶液中水的观察弛豫速率降低。向上述复合物溶液中添加硝酸盐会使酶对四氢叶酸和锰ADP的亲和力再增加5倍，并进一步降低弛豫速率，使其接近缺乏顺磁阳离子的酶和底物溶液的弛豫速率。
