Evidence for a singlet intermediate in catalysis by Escherichia coli DNA photolyase and evaluation of substrate binding determinants.
Escherichia coli DNA photolyase contains 1,5-dihydro-FAD (FADH2) plus 5,10-methenyl-tetrahydrofolate (5,10-CH+-H4folate). Both chromophores are fluorescent, and either can function as a sensitizer in catalysis. At 77 K separate fluorescence emission bands are observed for FADH2 (lambda max = 505 nm, shoulder at 540 nm) and 5,10-CH+-H4folate (lambda max = 465, 440 nm) whereas at 5 degrees C only a shoulder at 505 nm is attributable to FADH2. Formation of an enzyme-substrate complex with various dimer-containing oligothymidylates [UV-oligo(dT)n] quenches the fluorescence due to FADH2 at 5 degrees C or 77 K and also stabilizes FADH2 against air oxidation. The fluorescence of 5,10-CH+-H4folate is unaffected by substrate. Reduction of the pterin chromophore eliminates the chromophore's fluorescence but does not affect catalytic activity or the ability of substrate to quench FADH2 fluorescence. Quenching of FADH2 fluorescence is fully reversible upon dimer repair. The results are consistent with the proposal that the singlet state of FADH2 functions as an intermediate in catalysis. Fluorometric titrations indicate that the enzyme has a similar affinity for dimers in UV-oligo(dT)4 (KD = 2.5 X 10(-7) M, delta G = 8.4 kcal/mol at 5 degrees C) or UV-oligo(dT)6, except for dimers located at the unphosphorylated 3' end of the oligomers where binding is considerably weaker.(ABSTRACT TRUNCATED AT 250 WORDS)
Jordan
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SP
Department of Biological Chemistry, Hahnemann University School of Medicine, Philadelphia, Pennsylvania 19102.
Jorns
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MS
D002384:Catalysis; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives; D008190:Lyases / Q000378:metabolism*; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010084:Oxidation-Reduction; D010777:Photochemistry; D011740:Pyrimidine Dimers / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013763:Tetrahydrofolates; D014466:Ultraviolet Rays
D009838:Oligodeoxyribonucleotides; D011740:Pyrimidine Dimers; D013763:Tetrahydrofolates; C027903:oligo (dT); D005182:Flavin-Adenine Dinucleotide; C058805:1,5-dihydro-FAD; C018092:5,10-methenyltetrahydrofolate; D008190:Lyases; D004255:Deoxyribodipyrimidine Photo-Lyase