Transition State Analysis of Model and Enzymatic Prenylation Reactions

Abstract

Protein prenylation involves the attachment of C 15 (farnesyl) or C 20 (geranylgeranyl) groups to proteins and is catalyzed by a class of enzymes known as prenyltransferases. 1 The observation that inhibition of Ras farnesylation arrests the growth of tumor cells has been the motivating factor in developing inhibitors of prenyltransferases that can serve as anticancer drugs; currently several candidates are in Phase 3 clinical trials. 2 Mechanistic analysis of enzymatic reactions can provide insights that are potentially useful in drug design. Since enzymes must bind to the transition state (TS) with greater affinity than the ground state, molecules that mimic the structure of the TS will have the highest possible affinity for the enzyme. Enzyme inhibitors based on such principles can manifest extraordinary affinity and selectivity; 3 accordingly, we are interested in determining the TS structure for the reaction catalyzed by protein farnesyltransferase. Moreover, the detailed knowledge gained in these experiments should increase our understanding of how enzymes activate isoprenoid diphosphates for subsequent reaction. Such knowledge could be particularly useful for manipulating the reactivity of prenyltransferases and the closely related terpene cyclases for biotechnology purposes.At present, the most reliable method to determine TS structure is through the use of computational methods in conjunction with experimentally measured kinetic isotope effect (KIE) measurements. 4 While a large body of literature exists for KIE measurements performed on benzylic systems, reports for related allylic systems are sparse. Thus, as a prelude to enzymatic measurements, it was decided to investigate several model reactions, shown in Figure 1, first. Results from such experiments would provide KIE values for limiting associative (S N 2) and dissociative (S N 1) mechanisms and allow us to validate the computational methods that would be used in the subsequent determination of the enzymatic TS. For a model substrate, dimethylallyl chloride (1) was chosen. Solvolysis of 1 in benzyl alcohol (2) was used as a dissociative model while displacement with triphenylphosphine (5) was employed as an associative model. 5 Kinetic analysis of these reactions revealed that the solvolysis reaction was first order in 1 and zero order in 2 whereas the other reaction was first order in both 1 and 5. To measure the 13 C KIEs for the reaction, an NMR method was employed based on the work of Singleton 6 and others; that approach involves the integration of 13 C-NMR spectra obtained at natural abundance of reactant obtained prior to reaction and after substantial conversion. In the work reported here, the model reactions were performed, monitored by GC and terminated by vacuum distillation to recover the remaining starting material which was then derivatized by reaction with dimethylmalonate (7) and the resulting product (8) purified by flash chromatography. 13 C-NMR spectra were obtained and integrated using C-4 as an internal standard. 13 C KIEs we...

Structural plasticity in the human cytosolic sulfotransferase dimer and its role in substrate selectivity and catalysis

“…Therefore, current literature provides evidence for both S N 1 and S N 2 mechanisms. In reality, the actual mechanism may fall in between these two extremes, such as in the case of yeast protein farnesyltransferase, which exhibits a late transition state and is described as acting via a hybrid S N 1/S N 2 mechanism [74,75]. Further study of the SULT reaction mechanism is required before reaching this conclusion.…”

Understanding and Exploiting Protein Prenyltransferases

“…49 Kinetic isotope effect studies have suggested that the conformational rearrangement of phosphoisoprenoid prior to the chemical step is the rate‐limiting step of farnesylation 50. 51 Upon formation of a stable thioether bond, the pyrophosphate is released. The resulting negative charges are believed to be stabilized by conserved leucine and tyrosine residues in FTase and GGTase I. FTase needs Mg 2+ to stabilize the developing additional negative charge of the diphosphate group of FPP.…”

The Search for the Mechanism of the Reaction Catalyzed by Farnesyltransferase

“…However, in relation to the structure and nature of the transition state in the transfer of the farnesyl group from FPP to the cysteine sulfur atom of the CAAX substrate, very little has been done. In the only published computational attempt to analyze this elusive reaction step, [29] gas-phase structures of ethane thiolate and of a ten-carbon geranyldiphosphate were used to model this transfer reaction, suggesting C1 À O and C1 À S bond lengths of 1.69 and 3.70 , respectively. However, in this study the authors have not addressed the effect of zinc coordination sphere in the transition-state structure, a critical aspect for an accurate modeling of this reaction, particularly taking into consideration that it is a Zn-bound thiolate that acts as a nucleophile in this reaction, [21,30] and that both the reactant and the product in this step retain ZnÀS coordination.…”

“…[40] www.chemeurj.org Globally, these results direct to a concerted associative mechanism with a transition state that displays partial dissociative character as previously postulated from experimental studies. [20,21,29,30] …”