- Mechanistic studies of antibody-catalyzed pyrimidine dimer photocleavage
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An antibody elicited against the trans, syn uracil cyclobutane dimer hapten 1 catalyzes the light-dependent cleavage of uracil dimers 1 and 2 to the corresponding monomers 3 and 4. Kinetic analysis of the antibody-catalyzed reaction affords a value of k(cat)/K(M) = 1.7 x 103 M-1 min-1 for substrate 2, and comparison to the uncatalyzed reaction gives a rate acceleration of k(cat)/k(uncat) = 380. The wavelength dependence of the reaction and fluorescence quenching behavior suggest that a tryptophan residue is acting as a photosensitizer. The reaction mechanism was probed by measurement of secondary deuterium isotope effects. Substrates with selective deuterium substitutions in the cyclobutane ring were prepared, and isotope effects were measured by the method of interual competition using electrospray-ionization mass spectrometry to quantify the products. Kinetic isotope effects of (α-D)(V/K) = 1.11, 1.14, and 1.20 were observed for the 5,5'-, 6,6'-, and 5,5',6,6'-labeled substrates, respectively. These results are comparable to those observed in a similar study on the E. coli enzyme DNA photolyase and suggest that the reaction may proceed via a radical anion intermediate with concerted breakage of the 5,5' and 6,6' bonds. Alternatively the reaction may proceed via a mechanism in which the first bond is cleaved in a reversible fashion.
- Jacobsen, John R.,Cochran, Andrea G.,Stephans, James C.,King, David S.,Schultz, Peter G.
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- Nucleic acid bases studed by matrix isolation vibrational spectroscopy: uracil and deuterated uracils
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A vibrational assignment of the isolated uracil molecule is presented, based on i.r. and Raman spectra of uracil trapped in argon and nitrogen matrices and i.r. spectra of seven deuterated derivatives of uracil isolated in argon matrices.The observed spectra agree well with previous quantumm mechanical calculations.The relationships observed between the monomer bands, bands due to associated uracil in argon matrices and the spectrum of the pure solid lead to a revised vibrational assignment for uracil in the solid phase.As well as large shifts in the NH stretching and out-of-plane bending modes between the monomer and the hydrogen-bonded solid, many other bands show appreciable shifts as a consequence of the extensive mixing of the NH in-plane bending modes with the CH in-plane bending and ring stretching modes.The spectrum of the associated uracil species in matrices suggests that it is a cyclic dimer linked by C2=O...H-N1 hydrogen bonds.
- Barnes, A. J.,Stuckey, M. A.,Gall, L. Le
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p. 419 - 432
(2007/10/02)
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