Kinetics and mechanism of interaction of 10-propargyl-5,8-dideazafolate with thymidylate synthase.
Kinetics and mechanism of interaction of 10-propargyl-5,8-dideazafolate with thymidylate synthase.
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DOI：
10.1021/jm00154a009
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发表时间：
1986-04
影响因子：
7.3
通讯作者：
A. Pogolotti;P. Danenberg;D. Santi
中科院分区：
文献类型：
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作者：
A. Pogolotti;P. Danenberg;D. Santi
研究了干酪乳杆菌胸苷酸合酶 (TS) 与 10-炔丙基-5,8-二脱氮叶酸 (NPQ) 在 2'-脱氧尿苷酸 (dUMP) 存在下的相互作用。形成快速可逆的 dUMP-NPQ-酶复合物后，发生缓慢异构化，形成三元复合物，可以在硝酸纤维素膜上或通过凝胶过滤进行分离。可分离复合物的不寻常特征是其形成速率较慢 (t1/2 = 0.88 h) 和解离速率较慢 (t1/2 = 26.5 h)。三元复合物含有每摩尔二聚酶结合 2 摩尔 dUMP 和 2 摩尔 NPQ。 dUMP-NPQ-TS 复合物的紫外差异光谱显示出高波长最大值，这归因于结合时发生的酶和/或配体发色团的扰动。所提供的数据表明可分离三元复合物的形成涉及酶的催化硫醇基团对 dUMP 6 位的亲核攻击。这方面的证据如下：首先，嘧啶发色团在 265 nm 处的吸光度降低，其降低速度与可分离复合物的形成速度相同；其次，使用[6-3H]dUMP，在形成配合物时会产生大的逆α二级动力学同位素效应（kappa H/kappa T = 0.83），这与杂环6-碳的sp2到sp3重新杂化一致。用十二烷基硫酸钠 (NaDodSO4) 处理复合物会导致两个配体以未修饰的形式解离，这与复合物的拟议结构一致。当酶与 5-氟-2'-脱氧尿苷酸 (FdUMP) 和 NPQ 一起孵育时，也会形成可分离的三元复合物。有趣的是，FdUMP 从这些复合物中的解离是双相的，一半的结合核苷酸解离速度非常慢（t1/2 相当于 100 小时）。研究结果与 NPQ 作为抗癌剂的可能用途进行了讨论。
The interaction of Lactobacillus casei thymidylate synthase (TS) with 10-propargyl-5,8-dideazafolate (NPQ) in the presence of 2'-deoxyuridylate (dUMP) has been investigated. After formation of a rapidly reversible dUMP-NPQ-enzyme complex, a slow isomerization occurs to provide a ternary complex that can be isolated on nitrocellulose membranes or by gel filtration. Unusual features of the isolable complex are the slow rate by which it is formed (t1/2 = 0.88 h) and the slow rate at which it dissociates (t1/2 = 26.5 h). The ternary complexes contain 2 mol of dUMP and 2 mol of NPQ bound per mol of dimeric enzyme. Ultraviolet difference spectra of the dUMP-NPQ-TS complex shows a high wavelength maximum that has been attributed to perturbations of the enzyme and/or ligand chromophores that occur upon binding. Data are presented that suggest that the formation of the isolable ternary complex involves nucleophilic attack by a catalytic thiol group of the enzyme to the 6-position of dUMP. Evidence for this is as follows: first, there is a decrease in the absorbance of the pyrimidine chromophore at 265 nm that occurs at the same rate as the formation of the isolable complex; second, using [6-3H]dUMP there is a large, inverse alpha-secondary kinetic isotope effect (kappa H/kappa T = 0.83) upon formation of the complex that is in accord with sp2 to sp3 rehybridization of the 6-carbon of the heterocycle. Treatment of the complex with sodium dodecyl sulfate (NaDodSO4) results in the dissociation of both ligands in an unmodified form, which is consistent with proposed structure of the complex. Isolable ternary complexes are also formed when the enzyme is incubated with 5-fluoro-2'-deoxyuridylate (FdUMP) and NPQ. Interestingly, the dissociation of FdUMP from these complexes is biphasic, with one-half of the bound nucleotide dissociating at an exceedingly slow rate (t1/2 congruent to 100 h). The findings are discussed with relationship to the possible use of NPQ as an anticancer agent.