Photochemical studies and ultraviolet sensitization of Escherichia coli thymidylate kinase by various halogenated substrate analogs.

Abstract

The effect of 5-iodo-2'-deoxyuridine monophosphate (IdUMP), various 5-halogenated-5'-azido-2', 5' -dideoxyuridine derivatives, 2'-deoxy-6-azauridine (AzdUrd), and its halogenated analogs on the ultraviolet sensitization of Escherichia coli thymidylate kinase has been investigated. Only those compounds iodinated in position 5 enhance the rate of ultraviolet inactivation of this enzyme. However, 5'-azido nucleosides with iodo, bromo, chloro, or fluoro substituents in position 5 neither protect nor sensitize thymidylate kinase to ultraviolet inactivation. Thymidine 5'-monophosphate partially protects the enzyme against ultraviolet inactivation either in the presence or absence of ultraviolet-sensitizing iodinated analogs. Magnesium ion does not enhance the ultraviolet inactivation of thymidylate kinase by 5-iodinated nucleoside analogs. The kinatic data support an active site-directed enhancement of the enzyme to ultraviolet inactivation by 5-iodo-2'-deoxyuridine monophosphate, since the concentration of IdUMP required to attain 50% maximal enhancement is 0.24 mM which is in good agreement with its Ki of 0.18 mM. When either [125I]IdUMP or [2-14C]IdUMP was irradiated with the enzyme, both radioactivities were associated with the enzyme, however only with the 14C analog was the amount bound at half-saturation essentially equal to the amount required to inactivate the enzyme by 50%. These data support the hypothesis that the active entity in the enhancement by IdUMP of thymidylate kinase inactivation during ultraviolet irradiation is the uridylate free radical which is formed photochemically from IdUMP. Photochemical studies of 6-azauracil (AzUra), 2'-deoxy-6-azauridine, and 5-iodo-2'-deoxy-6-azauridine (IAzdUrd) were performed. Photolysis of IAzdUrd in the presence of a hydrogen donor yields AzdUrd which upon further photolysis yields the photohydrate. The photohydrate of AzdUrd when incubated in the dark at pH 5.2 is 90% converted back to AzdUrd, whereas the photohydrate of AzUra is only partially (20%) converted to AzUra. The rate of deiodination of IAzdUrd is 2.1-fold greater than that of IdUMP. Although the Ki of IdUMP and IAzdUrd is similar, the increased photosensitivity of the aza analog accounts for the much greater enhancement of ultraviolet inactivation of thymidylate kinase. The ability of a compound to enhance the ultraviolet inactivation of deoxythymidylate kinase is correlated with the potential of the compound to produce a free radical rather than a photohydrate when the enzyme-substrate analog complex is irradiated.