Mejillano M R, Jahansouz H, Matsunaga T O, Kenyon G L, Himes R H
Department of Biochemistry, University of Kansas, Lawrence 66045.
Biochemistry. 1989 Jun 13;28(12):5136-45. doi: 10.1021/bi00438a034.
N10-Formyltetrahydrofolate synthetase from bacteria and yeast catalyzes a slow formate-dependent ADP formation in the absence of H4folate. The synthesis of formyl phosphate by the enzyme was detected by trapping the intermediate as formyl hydroxamate. That the "formate kinase" activity was part of the catalytic center of N10-formyltetrahydrofolate synthetase was shown by demonstrating coordinate inactivation of the "kinase" and synthetase activities by heat and a sulfhydryl reagent, similar effects of monovalent cations, similar Km values for substrates, and similar Ki values for the inhibitor phosphonoacetaldehyde for both activities. The relative rates of the kinase activities for the bacterial and yeast enzymes are about 10(-4) and 4 x 10(-6) of their respective synthetase activities. These slow rates for the kinase reaction can be explained by the slow dissociation of ADP and formyl phosphate from the enzyme. This conclusion is supported by rapid-quench studies where a "burst" of ADP formation (6.4 s-1) was observed that is considerably faster than the steady-state rate (0.024 s-1). The demonstration of enzyme-bound products by a micropartition assay and the lack of a significant formate-stimulated exchange between ADP and ATP provide further evidence for the slow release of the products from the enzyme. The synthesis of N10-CHO-H4folate when H4folate was added to the E-formyl phosphate-ADP complex is also characterized by a "burst" of product formation. The rate of this burst phase at 5 degrees C occurs with a rate constant of 18 s-1 compared to 14 s-1 for the overall reaction at the same temperature. These results provide further evidence for formyl phosphate as an intermediate in the reaction and are consistent with the sequential mechanism of the normal catalytic pathway. Positional isotope exchange experiments using [beta,gamma-18O]ATP showed no evidence for exchange during turnover experiments in the presence of either H4folate or the competitive inhibitor pteroyltriglutamate. The absence of scrambling of the 18O label as observed by 31P NMR suggests that the central complex may impose restraints to limit free rotation of the P beta oxygens of the product ADP.
来自细菌和酵母的N10-甲酰四氢叶酸合成酶在没有四氢叶酸（H4folate）的情况下催化缓慢的依赖甲酸的ADP形成。通过将中间体捕获为甲酰异羟肟酸来检测该酶催化的甲酰磷酸的合成。“甲酸激酶”活性是N10-甲酰四氢叶酸合成酶催化中心的一部分，这一点通过热和巯基试剂对“激酶”和合成酶活性的协同失活、一价阳离子的类似作用、底物的类似Km值以及抑制剂膦酰乙醛对两种活性的类似Ki值得以证明。细菌和酵母酶的激酶活性的相对速率分别约为其各自合成酶活性的10^(-4)和4×10^(-6)。激酶反应的这些缓慢速率可以通过ADP和甲酰磷酸从酶上的缓慢解离来解释。这一结论得到了快速淬灭研究的支持，在该研究中观察到ADP形成的“爆发”（6.4 s^(-1)），其速度比稳态速率（0.024 s^(-1)）快得多。通过微分配测定法证明酶结合产物以及甲酸盐刺激的ADP与ATP之间缺乏显著交换，为产物从酶上的缓慢释放提供了进一步证据。当将H4folate添加到E-甲酰磷酸-ADP复合物中时，N10-CHO-H4folate的合成也以产物形成的“爆发”为特征。在5℃下，该爆发阶段的速率常数为18 s^(-1)，而在相同温度下整体反应的速率常数为14 s^(-1)。这些结果为甲酰磷酸作为反应中间体提供了进一步证据，并且与正常催化途径的顺序机制一致。使用[β,γ-18O]ATP进行的位置同位素交换实验表明，在存在H4folate或竞争性抑制剂蝶酰三谷氨酸的周转实验期间没有交换的证据。通过31P NMR观察到的18O标记没有重排，这表明中心复合物可能施加限制以限制产物ADP的Pβ氧的自由旋转。
