Evaluation of Isoprenoid Conformation in Solution and in the Active Site of Protein-Farnesyl Transferase Using Carbon-13 Labeling in Conjunction with Solution- and Solid-State NMR

Abstract

The enzyme protein-farnesyl transferase (FTase) catalyzes the farnesylation of the Ras protein and other key signal transduction proteins, using farnesyl diphosphate (FPP) as the prenyl source. Inhibitors of FTase are thus of great interest as potential novel anticancer agents. The design of such agents would be informed by a detailed knowledge of the solution conformation of FPP, as well as its conformation in the active site of FTase. Four bis-13 C-labeled derivatives of farnesol and geranylgeraniol have been synthesized and used to prepare the corresponding FPP and GGPP derivatives. The labeled farnesyl and geranylgeranyl derivatives 2-7 were utilized in conjunction with solution 13 C NMR to probe the conformation of the prenyl chain in a variety of different solvents. These studies, along with molecular dynamics simulations, demonstrate that the prenyl chain exists primarily in an extended conformation. Surprisingly, this preference for the extended conformation is solvent-insensitive; no significant change in conformation is seen with all six solvents investigated, including water. The [6,15-bis 13 C]FPP analogue 8 was complexed with mammalian FTase, and this complex was utilized in conjunction with rotational resonance MAS NMR to investigate the prenyl chain conformation when bound in the active site of this enzyme. The conformation determined from these experiments is in good agreement with the structure determined from crystallographic studies on the FPP-FTase complex. Thus, the isoprenyl chain of FPP exhibits a strong preference for an extended conformation, both in a variety of solvents of different polarities and in the active site of mammalian FTase.The mevalonate pathway, both the main trunk and its various branch points, has been the subject of intense pharmaceutical, biochemical, and chemical research activity. 1 The chemical mechanisms of steroid and terpene cationic cyclization reactions have been the subjects of long-standing and intensive research interest from bioorganic and biological chemists. [2][3][4][5] The recent

Computational and Conformational Evaluation of FTase Alternative Substrates: Insight into a Novel Enzyme Binding Pocket

“…e Carbon-13 NMR coupling constant previously determined from 5,15-bis-13 C-2E-farnesol (7). 12 Consistent with a trans conformational preference. f Values from the indicated bonds in the FPP in the experimentally determined FPP-FTase (1FT2) complex.…”

“…We have previously shown that NMR analysis of bis-13 C-labeled isoprenoids can provide insight into the conformational preferences of isoprenoids and thus confirm and extend computational and structural studies on these molecules. 11,12 In view of the intriguing biological activity of 2Z-GGPP, we synthesized the labeled molecules 4 and 5 (Figure 1). The synthetic routes to 4 and 5 are outlined in Schemes 1 and 2.…”

Sesquiterpene Synthases Cop4 and Cop6 from Coprinus cinereus: Catalytic Promiscuity and Cyclization of Farnesyl Pyrophosphate Geometric Isomers

“…FPP adopts an extended conformation in solution [63], but the active site of a terpene synthase must bind FPP in a helical conformation to facilitate cyclization. It is obvious that the conformation of the substrate in the active site controls the stereochemical course of the cyclization reaction.…”

Detection of nonsterol isoprenoids by HPLC–MS/MS

“…After the enzymatic syntheses, the samples were deproteinized using Microcon Centrifugal Filter Devices YM-10 (Millipore) according to the manufacturer’s protocol. GPP-d3, FPP-d3 and GGPP-d3 were prepared using the vinyl triflate methodology previously developed in the Gibbs laboratory [21;22]. Full details of the synthesis of these compounds will be published elsewhere.…”