COMPARATIVE-STUDIES ON S-ADENOSYL-L-METHIONINE BINDING-SITES OF PROTEIN N-METHYLTRANSFERASES, USING 8-AZIDO-S-ADENOSYL-L-METHIONINE AS PHOTOAFFINITY PROBE

Employing a photoaffinity labeling procedure with 8-azido-S-adenosyl-L -[methyl-H-3]methionine (8-N-3-Ado[methyl-H-3]Met), the binding sites for S-adenosyl-L-methionine (AdoMet) of three protein N-methyltransfer ases [AdoMet:myelin basic protein-arginine N-methyltransferase (EC2.1. 1.23); AdoMet:histone-arginin N-methyltransferase (EC2.1.1.23); and Ad oMet:cytochrome c-lysine N-methyltransferase (EC2.1.1.59)] have been i nvestigated. The incorporation of the photoaffinity label into the enz ymes upon W irradiation was highly specific. In order to define the Ad oMet binding sites, the photolabeled enzymes were sequentially digeste d with trypsin, chymotrypsin, and endoproteinase Glu-C. After each pro teolytic digestion, radiolabeled peptide from each enzyme was resolved on HPLC first by gradient elution and further purified by an isocrati c elution. Retention times of the purified radiolabeled peptides from the three enzymes from the corresponding proteolysis were significantl y different, indicating that their sizes and compositions were differe nt. Amino acid composition analysis of these peptides confirmed furthe r that the AdoMet binding sites of these protein N-methyltransferases are quite different.