The challenging task of identifying and studying protein function has been greatly aided by labeling proteins with reporter groups. Here, we present a strategy that utilizes an enzyme that labels a four-residue sequence appended onto the C terminus of a protein, with an alkyne-containing substrate. By using a bio-orthogonal cycloaddition reaction, a fluorophore that carried an azide moiety was then covalently coupled to the alkyne appended on the protein. FRET was used to calculate a Förster (R) distance of 40 A between the eGFP chromophore and the newly appended Texas Red fluorophore. This experimental value is in good agreement with the predicted R value determined by using molecular modeling. The small recognition tag, the high specificity of the enzyme, and the orthogonal nature of the derivatization reaction will make this approach highly useful in protein chemistry.
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...
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