Probing the S-adenosylmethionine-binding site of rat guanidinoacetate methyltransferase. Effect of site-directed mutagenesis of residues that are conserved across mammalian non-nucleic acid methyltransferases.

Most mammalian non-nucleic acid methyltransferases share three sequence motifs. To gain insight into the S-adenosyl-methionine (AdoMet)-binding site of guanidinoacetate methyltransferase, we mutated several conserved residues that are found in or near motifs I and II. Conversion of either of two glycine residues of motif I (Gly67 and Gly69) to an alanine resulted in an inactive enzyme. These enzymes, although having UV absorption, fluorescence and far-UV CD spectra virtually identical with those of the wild-type enzyme, seem to be conformationally different from the wild-type enzyme as judged by near-UV CD spectra and the extent of urea denaturation, and are apparently not capable of binding AdoMet. Mutation of Tyr136 of motif II to a valine resulted in a decrease in Kcat/Km values for substrates. Changing this residue to a phenylalanine caused only a minor change in Kcat/Km for AdoMet. This suggests that the aromatic side chain stabilizes the binding of AdoMet. Mutagenic changes of Glu89, which is the residue corresponding to the conserved acidic residue on the C-terminal side of motif I, indicated its contribution to AdoMet binding. These results are consistent with the idea that both motifs I and II are crucial in forming the AdoMet binding site of guanidinoacetate methyltransferase.