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.
Hamahata
A
A
Department of Biochemistry, Toyama Medical and Pharmaceutical University Faculty of Medicine, Japan.
Takata
Y
Y
Gomi
T
T
Fujioka
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D017124:Conserved Sequence; D005998:Glycine / Q000031:analogs & derivatives* / Q000378:metabolism; D050937:Guanidinoacetate N-Methyltransferase; D007700:Kinetics; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D051381:Rats; D012436:S-Adenosylmethionine / Q000378:metabolism*
D012436:S-Adenosylmethionine; D008780:Methyltransferases; C494508:Gamt protein, rat; D050937:Guanidinoacetate N-Methyltransferase; C004946:glycocyamine; D005998:Glycine