Molecular mechanics calculations on cis ([Pt(NH3)2{d(GpG)}] adducts in two oligonucleotide duplexes
Molecular mechanics calculations on cis ([Pt(NH3)2{d(GpG)}] adducts in two oligonucleotide duplexes
复制标题
两个寡核苷酸双链体中顺式 ([Pt(NH3)2{d(GpG)}] 加合物的分子力学计算
DOI：
10.1021/ja00299a055
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发表时间：
1985
影响因子：
15
通讯作者：
G. Quigley
中科院分区：
文献类型：
--
作者：
J. Kozelka;G. Petsko;S. Lippard;G. Quigley
测定混合物包括0.1M马来酸钠，pH 6.3，10 mM氯化镁，[4-14C]焦磷酸异戊烯(10-100微米)，抑制剂和异构酶(0.5单位)，总体积为0.25毫升。通过添加酶来启动检测。在37℃下反应6min后，加入15pl的5-N-HCl，并测定产物的生成量。8在所有情况下，K值由1/v与1/[IPP]曲线的斜率曲线确定。使用三种抑制剂浓度和四种底物浓度。用已发表的方法合成了6，2-二甲氨基)乙基磷酸酯，并在pH值为8.9的条件下进行了纸层析纯化。3-溴-3-丁烯醇按文献10合成，异戊烯基磷酸酯按文献11合成，焦磷酸异戊烯酯按磷酸二苯酯偶联法合成。12用上述方法合成所有其他焦磷酸单酯，并用QAE Sephadex层析纯化，用线性三乙基碳酸氢铵梯度从0.0到0.6M。在pH 3.5和/或8.9的条件下，用核磁共振和纸电泳法测定所有焦磷酸单酯的纯度。纸层析和纸层析样品用钼酸铵喷雾显影。用磷酸盐分析法测定了13个用于酶测定的底物和抑制剂样品的浓度。14在酶-抑制物复合体形成后发生缓慢事件。3与IPPI孵育更直接地显示了抑制作用的时间依赖性。每隔一段时间，抽出等份并测定酶活性。结果如图1b所示。在所测试的所有缓蚀剂浓度下，催化活性的丧失是一个一级过程。在测试的浓度范围内，kobsi与酶浓度成正比。没有看到饱和的证据。图1b还显示底物降低了失活速率，表明3可能与活化位相互作用。当IPPI与不含镁离子的3在5 mM EDTA存在下孵育时，不出现抑制作用。因此，这种抑制是依赖于镁离子的，催化过程也是如此。对3的几个类似物也进行了研究。所有受试化合物都是竞争性抑制物，但没有观察到时间依赖性抑制作用。表I列出了K值。为了确定IPPI与3的反应是否可逆，将6.67微米3加入到分析缓冲液中的酶(11个单位)中，总体积为150微米。37℃10分钟后，通过等份分析，确定该酶失活了95%。然后对反应混合物进行快速凝胶过滤，以去除多余的抑制剂。过滤后未检测到任何酶活性，凝胶过滤90min后也未检测到任何活性。将酶和缓蚀剂混合后，在10 S以内进行凝胶过滤，进行平行实验。过滤后即刻酶活力恢复到原来的67%，90分钟后恢复到原来的50%。结果表明，3对异戊烯焦磷酸异构酶的抑制具有时间依赖性。从到目前为止的数据，还不能确定酶抑制剂复合体的解离是否非常缓慢(解离的t1/2为10h)，也不能确定是否发生了不可逆酶的修饰。通过将甲基从3转移到活性部位的亲核试剂上，可能发生不可逆转的失活。我们认为，3对异戊烯基焦磷酸异构酶的抑制为碳正离子机制提供了强有力的证据。现在有几个例子表明，碳原子的取代会导致…
“Assay mixtures contained 0.1 M sodium maleate pH 6.3, 10 mM MgCl2,[4-14C] isopentenyl pyrophosphate (10-100 µ), inhibitor, and isomerase (0.5 units) in 0.25-mL total volume. Assays were initiated by addition of enzyme. After 6 min at 37 C, 15 pL of 5 N HC1 was added and the amount of product formed was determined. 8 In all cases, K, values were determined from theslope replots of 1/v vs. 1/[IPP] plots. Three inhibitor concentrations and four substrate con-centrations were used. 6 2-(Dimethylamino) ethyl phosphate was synthesized by a published procedure9 and purified by paper electropho-resis at pH 8.9. 3-Bromo-3-butenol was synthesized according to ref 10. Isopentenyl phosphate was synthesized according to ref 11. Iso-pentenyl pyrophosphate was synthesized by using the diphenyl phos-phorochloridate coupling method. 12 All other pyrophosphate mono-esters were synthesized by the above procedures and purified by chromatography on QAE Sephadex using a linear triethylammonium bicarbonate gradient from 0.0 to 0.6 M. The purity of all phosphate and pyrophosphate monoesters was determined by NMR and paper electrophoresis at pH 3.5 and/or 8.9. Samples from paper electropho-resis and paper chromatography were visualized with ammonium molybdate spray. 13 Concentrations of substrate and inhibitor samples for use in enzyme assays were determined by phosphateanalysis. 14 a slow event occurs subsequent to formation of enzyme-inhibitor complex. Time dependence of the inhibition was demonstrated more directly by incubating 3 with IPPI. At intervals, aliquots were withdrawn and assayed for enzyme activity. Results are shown in Figure lb. Loss of catalytic activity is a first-order-order process at all inhibitor concentrations tested. Over the range of concentrations tested, kobsi is proportional to enzyme concentration. No evidence for saturation was seen. Figure lb also shows that substrate reduces the rate of inactivation, indicating that 3 probably interacts with the activesite. When IPPI was incubated with 3 without Mg2+ in the presence of 5 mM EDTA, no inhibition occurred. The inhibition is, therefore, Mg2+ dependent, as is the catalytic process. Several analogues of 3 were also investigated. All compounds tested were competitive inhibitors, but no timedependent inhibition was observed. The K, values are listed in Table I. To determine whether the reaction of IPPI with 3 is reversible, 6.67 µ 3 was added to enzyme (11 units) in the assay buffer in a total volume of 150 µ. After 10 min at 37 C, it was determined, by assaying an aliquot, that the enzyme was> 95% inactivated. The reaction mixture was then subjected to rapid gel filtration4 to remove excess inhibitor. No enzyme activity was detected after filtration nor was any activity detected 90 min after gel filtration. A parallel experiment was carried out in which enzyme and inhibitor were mixed and subjected to gel filtration within 10 s after mixing. Immediately after filtration theenzyme had 67% of its original activity and 50% after 90 min. The result presented hereshows that inhibition of isopentenyl pyrophosphate isomerase by 3 is time dependent. From the data available so far, it cannot be determined whether dissociation of the enzyme inhibitor complex is extremely slow (t1/2 for disso-ciation> 10 h) or whether irreversible modification of the enzyme occurs. Possibly irreversible inactivation could occur by methyl transfer from 3 to a nucleophile at the active site. We believe that inhibition of isopentenyl pyrophosphate isom-erase by 3 provides strong evidence for a carbonium ion mecha-nism. There are now several examples in which substitution of a carbon atom, which …