Photoaffinity labelling of the 2-oxoglutarate binding site of prolyl 4-hydroxylase with 5-azidopyridine-2-carboxylic acid.

The synthesis of the photoaffinity label 5-azidopyridine-2-carboxylic acid is described. The 2-oxoglutarate analogue photoaffinity label is a competitive inhibitor with respect to 2-oxoglutarate with a Ki value of 9 X 10(-3) M. Upon ultraviolet irradiation, 5-azidopyridine-2-carboxylic acid inactivated prolyl 4-hydroxylase irreversibly by up to 50%. The extent of inactivation depended on the 5-azidopyridine-2-carboxylic acid concentration and the irradiation time. Inactivation was prevented in the presence of an excess of 2-oxoglutarate. It is concluded that the 5-azidopyridine-2-carboxylic acid became covalently bound to the alpha subunit of prolyl 4-hydroxylase, as the alpha subunit of the photoaffinity labelled enzyme had a decreased electrophoretic mobility in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate.