157807-83-5Relevant articles and documents
Incorporation of fluorotyrosines into ribonucleotide reductase using an evolved, polyspecific aminoacyl-tRNA synthetase
Minnihan, Ellen C.,Young, Douglas D.,Schultz, Peter G.,Stubbe, Joanne
, p. 15942 - 15945 (2011)
Tyrosyl radicals (Y?s) are prevalent in biological catalysis and are formed under physiological conditions by the coupled loss of both a proton and an electron. Fluorotyrosines (FnYs, n = 1-4) are promising tools for studying the mechanism of Y? formation and reactivity, as their pK a values and peak potentials span four units and 300 mV, respectively, between pH 6 and 10. In this manuscript, we present the directed evolution of aminoacyl-tRNA synthetases (aaRSs) for 2,3,5-trifluorotyrosine (2,3,5-F3Y) and demonstrate their ability to charge an orthogonal tRNA with a series of FnYs while maintaining high specificity over Y. An evolved aaRS is then used to incorporate FnYs site-specifically into the two subunits (α2 and β2) of Escherichia coli class Ia ribonucleotide reductase (RNR), an enzyme that employs stable and transient Y?s to mediate long-range, reversible radical hopping during catalysis. Each of four conserved Ys in RNR is replaced with FnY(s), and the resulting proteins are isolated in good yields. FnYs incorporated at position 122 of β2, the site of a stable Y? in wild-type RNR, generate long-lived FnY?s that are characterized by electron paramagnetic resonance (EPR) spectroscopy. Furthermore, we demonstrate that the radical pathway in the mutant Y122(2,3,5)F3Y-β2 is energetically and/or conformationally modulated in such a way that the enzyme retains its activity but a new on-pathway Y? can accumulate. The distinct EPR properties of the 2,3,5-F3Y? facilitate spectral subtractions that make detection and identification of new Y?s straightforward.
Kinetic analysis of a protein tyrosine kinase reaction transition state in the forward and reverse directions
Kim, Kyonghee,Cole, Philip A.
, p. 6851 - 6858 (2007/10/03)
Protein tyrosine kinases catalyze the transfer of the γ-phosphoryl group from ATP to tyrosine residues in proteins and are important enzymes in cell signal transduction. We have investigated the catalytic phosphoryl transfer transition state of a protein tyrosine kinase reaction catalyzed by Csk by analyzing a series of fluorotyrosine-containing peptide substrates. It was established for five such fluorotyrosine-containing peptide substrates that there is good agreement between the tyrosine analogue phenol pK(a) and the ionizable group responsible for the basic limb of a pH rate profile analysis. This indicates that the substrate tyrosine phenol must be neutral to be enzymatically active. Taken together with previous data indicating a small β(nucleophile) coefficient (0-0.1), these results strongly support a dissociative transition state for phosphoryl transfer. In addition, the β(leaving group) coefficient was measured for the reverse protein tyrosine kinase reaction and shown to be -0.3. This value is in good agreement with a previously reported nonenzymatic model phosphoryl transfer reaction carried out under acidic conditions (pH 4) and is most readily explained by a transition state with significant proton transfer to the departing phenol.
