Rat guanidinoacetate methyltransferase. Effect of site-directed alteration of an aspartic acid residue that is conserved across most mammalian S-adenosylmethionine-dependent methyltransferases.
Most mammalian S-adenosylmethionine (AdoMet)-dependent methyltransferases have a conserved aspartate residue in a sequence oDso (o denotes a hydrophobic amino acid and s denotes a small neutral amino acid). Rat guanidinoacetate methyltransferase has two aspartate residues (Asp-129 and Asp-134) conforming to the motif in close proximity to Tyr-136 that is photoaffinity-labeled by AdoMet (Takata, Y., and Fujioka, M. (1992) Biochemistry 31, 4369-4374). In order to investigate the role of these residues, we prepared variant forms of the enzyme by oligonucleotide-directed mutagenesis. Conversion of Asp-129 to asparagine or alanine resulted in a functional enzyme. Alteration of Asp-134 to glutamate (D134E) and asparagine (D134N) decreased activity, and replacement with alanine (D134A) led to inactivation. Decreases of 3- and 120-fold were found for kcat values of D134E and D134N, respectively. The Km values of D134E for AdoMet and those for guanidinoacetate were increased about 160- and 80-fold over the respective values of wild type. The corresponding increases in D134N were 800- and 50-fold, respectively. Conservative changes of the residues flanking Asp-134 had little effect on activity. Guanidinoacetate methyltransferase obeys an ordered Bi Bi mechanism in which AdoMet binds first. Thus, the large decreases in kcat/Km values for AdoMet indicate that Asp-134 is crucial for binding AdoMet. Spectroscopic studies indicated that the amino acid substitutions of Asp-134 resulted in no significant changes in the secondary and tertiary structures, and urea denaturation experiments showed that the altered enzymes were not destabilized.
Takata
Y
Y
Department of Biochemistry, Toyama Medical and Pharmaceutical University Faculty of Medicine, Japan.
Konishi
K
K
Gomi
T
T
Fujioka
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D017124:Conserved Sequence; D018076:DNA, Complementary; D050937:Guanidinoacetate N-Methyltransferase; D007700:Kinetics; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D051381:Rats; D012436:S-Adenosylmethionine / Q000378:metabolism*; D014508:Urea
D018076:DNA, Complementary; D001224:Aspartic Acid; D012436:S-Adenosylmethionine; D014508:Urea; D008780:Methyltransferases; C494508:Gamt protein, rat; D050937:Guanidinoacetate N-Methyltransferase