Transition State Analysis of Model and Enzymatic Prenylation Reactions

To obtain a transition state (TS) structure for an enzyme-catalyzed prenylation reaction, SN1 and SN2 model substitution reactions with dimethylallyl chloride were first studied. 13C Kinetic isotope effects (KIEs) for the model reactions were measured by a natural abundance NMR method and used to validate the computational methods that would be used in the subsequent determination of the enzymatic TS structure. Using a primary 13C KIE and a secondary 2H KIE measured via mass spectrometry, a TS structure for the enzyme-catalyzed reaction was computed; a density functional level of electronic structure theory using the mPW1N functional in combination with the 6-31+G(d) basis set was employed for those calculations. That structure has a C−O bond length of 1.69 Å and a C−S bond length of 3.70 Å. While the former bond length is similar to that for a nonenzymatic SN2 reaction, the latter is considerably (0.90 Å) longer, indicating that the enzyme effects catalysis via an “exploded” TS structure.为获取酶催化异戊烯基化反应的过渡态（Transition State, TS）结构，研究人员率先针对以氯化二甲基烯丙基为底物的SN1与SN2型模型取代反应开展研究。研究人员采用天然丰度核磁共振（Nuclear Magnetic Resonance, NMR）法测定了该模型反应的13C动力学同位素效应（Kinetic Isotope Effects, KIEs），并以此验证后续用于确定酶促过渡态结构的计算方法。依托经质谱法测得的一级13C动力学同位素效应与二级2H动力学同位素效应，研究人员计算得到了该酶催化反应的过渡态结构；此类计算采用结合6-31+G(d)基组的mPW1N泛函的密度泛函电子结构理论方法。该过渡态结构的C−O键长为1.69 Å，C−S键长为3.70 Å。尽管前者键长与非酶促SN2反应的键长相近，但后者显著更长（差值达0.90 Å），这表明该酶通过“扩张型”过渡态结构发挥催化作用。