5-10-methenyltetrahydrofolate and 5-11-methenyltetrahydrohomofolate

Escherichia coli DNA photolyase mediates photorepair of pyrimidine dimers occurring in UV-damaged DNA. The enzyme contains two chromophores, 1,5-dihydroflavin adenine dinucleotide (FADH2) and 5,10-methenyltetrahydrofolylpolyglutamate (MTHF). To define the roles of the two chromophores in the photochemical reaction(s) resulting in DNA repair and the effect of DNA structure on the photocatalytic step, we determined the absolute action spectra of the enzyme containing only FADH2 (E-FADH2) or both chromophores (E-FADH2-MTHF), with double- and single-stranded substrates and with substrates of different sequences in the immediate vicinity of the thymine dimer. We found that the shape of the action spectrum of E-FADH2 matches that of the absorption spectrum with a quantum yield phi (FADH2) = 0.69. The action spectrum of E-FADH2-MTHF is also in a fairly good agreement with the absorption spectrum with phi (FADH2-MTHF) = 0.59. From these values and from the previously established properties of the two chromophores, we propose that MTHF transfers energy to FADH2 with a quantum yield of phi epsilon T = 0.8 and that 1FADH2 singlet transfers an electron to or from the dimer with a quantum yield phi ET = 0.69. The chemical nature of the chromophores did not change after several catalytic cycles. The enzyme repaired a thymine dimer in five different sequence contexts with the same efficiency. Similarly, single- and double-stranded DNAs were repaired with the same overall quantum yield.

Topics: Base Sequence; Deoxyribodipyrimidine Photo-Lyase; DNA Repair; Escherichia coli; Flavin-Adenine Dinucleotide; Folic Acid; Molecular Sequence Data; Protein Denaturation; Pyrimidine Dimers; Spectrophotometry, Ultraviolet; Substrate Specificity; Tetrahydrofolates

The formyl-methenyl-methylenetetrahydrofolate synthetase from chicken liver catalyzes the formation of the 10-formyl- and 5,10-methenyltetrahydrofolate cofactors via three enzymatic activities. In this report we define the kinetic relationships between the activities of this trifunctional protein. An investigation of the time course for 10-formyl cofactor synthesis by computer modeling indicates that commencing with tetrahydropteroyltriglutamate, the activities of the synthetase/cyclohydrolase couple act as separate enzymic species. In contrast, 10-formyl cofactor formation from the 5,10-methylene cofactor utilizing the dehydrogenase/cyclohydrolase couple is described by a single or interactive site model that partitions the 5,10-methenyl intermediate primarily (85%) to the 10-formyl product. An unusual characteristic of the latter coupled activities is the negligible cyclohydrolase activity toward exogenous 5,10-methenyl cofactor, which serves as substrate in the individual activity assay. This is based on (1) competitive inhibition by 5,11-methenyltetrahydrohomofolate against the 5,10-methenyl derivative in the cyclohydrolase-catalyzed hydrolysis but the absence of such inhibition in the dehydrogenase/cyclohydrolase couple and (2) a pulse-chase experiment showing the failure of chase 5,10-methenyl cofactor to dilute the 10-formyl product derived from the coupled activities. The result of this coupling is to minimize the concentration of the 5,10-methenyl species, consistent with its noninvolvement in de novo purine biosynthesis.

Topics: Aminohydrolases; Animals; Chickens; Folic Acid; Formate-Tetrahydrofolate Ligase; Kinetics; Leucovorin; Ligases; Liver; Mathematics; Methenyltetrahydrofolate Cyclohydrolase; Methylenetetrahydrofolate Dehydrogenase (NADP); Multienzyme Complexes; Oxidoreductases; Tetrahydrofolates; Time Factors