The structural features of S-adenosyl- l-methionine (SAM) 3 3 Abbreviations used: SAM, S-adenosyl- l-methionine; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; SAH, S-adenosylhomocysteine; NAc, N-α-acetyl; MAT, methionine adenosyltransferase; COMT, catechol- O-methyltransferase; DTT, dithiothreitol; BSA, bovine serum albumin; TCA, trichloroacetic acid; MTA, 5′-deoxy-5′-methylthioadenosine; SAE, S-adenosyl- l-ethionine. required for optimal binding to a nucleolar 2′- O-methyltransferase were elucidated using various analogs of SAM with modifications of the amino acid, sugar, sulfonium center, and base portions of the molecule. Equilibrium binding constants for SAM and each analog were determined by a nitrocellulose filter binding assay. To ensure the chiral and chemical purity of the 3H-labeled SAM used in the binding experiments, a cation-exchange HPLC procedure was developed to separate degradation products of SAM such as adenine and 5′-deoxy-5′-methylthioadenosine, as well as to separate the ( S,S)-SAM from the biologically inactive ( R,S)-SAM stereoisomer. Results from these studies demonstrated that S-adenosyl- l-homocysteine, a product of the methyltransferase reaction, bound equally as well as ( S,S)-SAM, indicating that neither the charge nor the methyl group at the sulfonium center of ( S,S)-SAM is essential for maximal binding. Other modifications of the sulfonium center demonstrated that a sulfur to carbon atom replacement had little effect on binding affinity, whereas substituting an ethyl group for the methyl group greatly reduced the binding affinity. In addition, the chirality at the sulfonium center was important. The naturally occurring S-chiral form had a 10-fold higher binding affinity than the R-chiral form. No significant stereospecificity was observed relative to the chiral α-carbon of the methionine moiety in SAM. The α-amino group of methionine and the 6-amino group of adenine were both required for maximal binding, while the loss of the 2′-hydroxyl group on the ribose moiety was not. Taken together, these results defined some of the specific geometric and functional group requirements which affect the specificity of interaction between S-adenosyl- l-methionine and the nucleolar 2′- O-methyltransferase.