title: Photoreactivating enzyme from Escherichia coli: isolated enzyme lacks absorption in its actinic wavelength region and its ribonucleic acid cofactor is partially double stranded when associated with apoprotein.
abstract: Isolated photoreactivating enzyme (PRE) from Escherichia coli exhibits some optical density at wavelengths greater than 300 nm. After correcting for the effects of light scattering, however, we find no true absorption in the spectral region that is required for enzymatic activity (320-450 nm). At shorter wavelengths, there is an absorption maximum near 260 nm that is due primarily to an RNA cofactor. Heating to 60 degrees C and subsequently cooling to 4 degrees C release the RNA cofactor from association with apoprotein and result in hyperchromicity. Circular dichroism indicates that the RNA associated with native enzyme is partially double stranded. At low ionic strength (mu = 0.01), heating to 15 degrees C or protease treatment at 4 degrees C results in irreversible loss of part of the double strandedness. We show that the difference spectrum at 4 degrees C between the absorption spectra of native enzyme and heat-treated enzyme can be fit by a superposition of reference spectra for denaturation of A-U and G-C base pairs derived from model polynucleotides. The coefficients of the linear combination of reference spectra were used to calculate the fraction of A-U and G-C base pairs. We find that both A-U and G-C base pairs are present in equal concentrations and that about 20% are in a double-stranded conformation in the native enzyme.
mesh_terms: D001482:Base Composition; D002942:Circular Dichroism; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006358:Hot Temperature; D008190:Lyases / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D010447:Peptide Hydrolases; D011487:Protein Conformation; D011740:Pyrimidine Dimers / Q000378:metabolism; D012329:RNA, Bacterial / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000378:metabolism*; D013053:Spectrophotometry
chemical_list: D011740:Pyrimidine Dimers; D012329:RNA, Bacterial; D012330:RNA, Double-Stranded; D010447:Peptide Hydrolases; D008190:Lyases; D004255:Deoxyribodipyrimidine Photo-Lyase