Photoenzymatic Repair of Ultraviolet Damage in DNA ii. Formation of an enzyme-substrate complex

The photoenzyme from bakers' yeast which repairs ultravioletinactivated transforming DNA is mechanically bound to ultraviolet-irradiated DNA in the dark, but not to unirradiated DNA. In the bound condition it is stabilized against inactivation by heat and heavy metals. Both the mechanical binding and stabilization are eliminated by illumination. These observations are consistent with the reaction scheme suggested by kinetic studies, in which the enzyme combines with the ultraviolet lesions in DNA and the complex absorbs light, producing repair and subsequent liberation of the enzyme. The approximately exponential decrease of heat stabilization during illumination gives the first order rate constant for the light-dependent step at the corresponding light intensity. This quantity in turn sets limits on the possible magnitude of the molar absorption coefficient of the enzymesubstrate complex and on the quantum yield of the process. I N T R O D U C T I O N The preceding paper (1) presented evidence that repair of ultraviolet damage to DNA by the yeast photoreactivating enzyme (YPRE) follows MichaelisMenten kinetics. In the reaction scheme conventionally associated with these kinetics, the enzyme first combines with its substrate (here, the ultravioletinduced lesions in DNA) and the resulting complex undergoes a first order reaction which yields the product (repaired DNA) and free enzyme (2): E + S~viE S ~ E + P Kinetic evidence points to the second reaction as the light-requiring step. If this picture is correct, the enzyme-substrate complex is a stable compound in the dark. I t should therefore be possible to demonstrate its existence by gentle fractionation procedures capable of separating unbound enzyme from