3'-O-(4-benzoyl)benzoylcytidine 5'-triphosphate. A substrate and photoaffinity label for CMP-N-acetylneuraminic acid synthetase

A photoreactive, radiolabeled pyrimidine nucleotide, 3'-O-(4-benzoyl)benzoylcytidine 5'-triphosphate was synthesized from benzoylbenzoic acid and radiolabeled CTP. Benzoylbenzoyl-[5-3H]CTP could substitute for CTP, in an enzymatic reaction with N-acetylneuraminic acid catalyzed by Escherichia coli or rat liver CMP-NeuAc synthetase, to yield radiolabeled benzoyl-benzoyl-CMP-NeuAc. E. coli CMP-NeuAc synthetase could be specifically radiolabeled using benzoylbenzoyl-[alpha-32P]CTP as a photoaffinity label. This specific covalent binding occurred using enzyme preparations of different degrees of purity. These results suggest that benzoylbenzoic acid derivatives of pyrimidines should be of general use in the identification and active site mapping of pyrimidine-requiring proteins and enzymes. These include glycosyltransferases, sugar nucleotide synthetases, and transporters, and enzymes participating in the conjugation of bile acids and biosynthesis of nucleic acids and choline nucleotides.