Analysis of photoenzymatic repair of UV lesions in DNA by single light flashes V. Determination of the reaction-rate constants in E. coli cells

Abstract

It was shown earlier that the use of intense light flashes permits analyzing in greater detail the reaction steps involved in photoenzymatic repair (PER). This paper describes experiments in which the reaction-rate constants for the formation of enzyme-substrate complexes ( k 1) and dark dissociation of the complexes ( k 2), and the photolytic constant ( k p ) for the light-dependent repair step have been determined in E. coli B s−1. All three reactions are heterogeneous with respect to these constants. Presumably this reflects the diversity of pyrimidine dimers in irradiated DNA, all of which serve as substrate for photoreactivating enzyme (PRE); the following values are weighted averages. At room temperature, k 1 equals 1.8·10 −3 cell volumes·molecules −1·sec −1, which for a cell volume of 10 −15 liter corresponds to 1.1·10 6 liter·mole −1·sec −1. For k 2, values ranging from 1.9·10 −3 to 1.3·10 −2 sec −1 have been obtained in different experimental approaches, depending on which criteria were used. k p has been determined under conditions where all of the substrate is complexed with photoreactivating enzyme at the beginning of the illumination. For the wavelengths 385, 366, and 355 nm k p values of, respectively, 1.75·10 −3, 1.37·10 −3, and 1.13·10 −3 mm 2·erg −1, have been obtained. From the values the product of ϵ (the molar extinction coefficient of the enzyme-substrate complexes) and Φ (the quantum yield of the photolytic reaction) can be calculated. At the most effective wavelength (385 nm), ϵΦ equals 2.4·10 4 liter·mole −1·cm −1, indicating that both ϵ and Φ must be high. ϵ must be ⩾2.4·100 4 lliter·mole −1·cm −1, since Φ cannot exceed 1; Φ is probably between 10 −1 and 1, since ϵ values >10 5 are unlikely. k 1 and k 2 show positive temperature dependence in the range 5 to 37°, corresponding to activation energies of 11 kcal·mole −1 and about 4.5 kcal·mole −1 for the respective steps, while k p was earlier shown to be essentially temperature-independent in this range.