Analysis of photoenzymatic repair of UV lesions in DNA by single light flashes VI. Rate constants for enzyme-substrate binding in vitro between yeast photoreactivating enzyme and ultraviolet lesions in Haemophilus transforming DNA

Abstract

Rate constants k 1 for formation, and k 2 for dissociation of the enzyme-substrate complexes of yeast photoreactivating enzyme and ultraviolet lesions in Haempphilus influenzae transforming DNA, have been measured as functions of temperature, pH and ionic strength in vitro. The measurements employed intense light flashes which cause repair of essentially all lesions complexed, to determine the number of complexes existing at any moment. Absolute concentrations of substrate were referred to the known numbers of pyrimidine dimers in irradiated DNA, while enzyme concentrations were determined by titration against substrate in a concentration range where the binding was essentially complete. The results reported permit a choice of optimum conditions for photoreactivation under various conditions of illumination. UV lesions are heterogeneous with respect to both formation and dissociation of complexes, and the values determined for the constants are weighted averages. The two rate constants depend on temperature as described by the Arrhenius expression with activation energies of about 9.3 and 5.5 kcal·mole −1, respectively. This particular difference between the two activation energies indicates that the binding energy in the complex is small. Both constants increase with pH over the range 6.0–7.7, but their ratio, which governs the equilibrium complexing in the dark, is higher at the lower pH. k 1 depends critically on the salt concentration, the position of the sharp maximum varying with the particular ions present, while k 2 increases generally with salt concentration over the range studied. Simple empirical expressions have been found which quantitatively describe the behavior of both constants as a function of ionic strength.