Photoaffinity cross-linking of TaqI restriction endonuclease using an aryl azide linked to the phosphate backbone

Abstract

In an effort to identify amino acid (aa) residues near the active site of TaqI restriction endonuclease (ENase), a sequence-specific photoaffinity reagent was designed. This reagent exploits the finding that modification of the Rp oxygen of the scissile phosphate does not interfere with substrate binding. The TpCGA phosphate was substituted with an Rp phosphorothioate group to direct the placement of the heterobifunctional reagent p-azidophenacyl bromide. TaqI bound the photoaffinity reagent specifically and formed a covalent adduct with the ENase in the presence of UV light. The modified aa was identified as Tyr161. This aa was changed to Phe by site-directed mutagenesis, and the resulting Y161F mutant was characterized. Removal of the Tyr161 hydroxyl group lowered both the kcat and the Km fivefold, indicating that, while this aa may be near the scissile phosphate, it is not critically required for catalysis.