Recombinant expression of rat glycine N-methyltransferase and evidence for contribution of N-terminal acetylation to co-operative binding of S-adenosylmethionine.
An expression vector was constructed that produced rat glycine N-methyltransferase in Escherichia coli. Recombinant glycine N-methyltransferase was purified to homogeneity by DEAE-cellulose and gel-filtration chromatography, with a yield of more than 80 mg of pure enzyme from a 1 litre culture. HPLC of tryptic peptides and analysis of isolated peptides showed that the recombinant enzyme was structurally identical with the liver enzyme except for the absence of N-terminal blocking. The alpha-amino group of rat glycine N-methyltransferase is blocked by acetylation [Ogawa, Konishi, Takata, Nakashima and Fujioka (1987) Eur. J. Biochem. 168, 141-151]. In contrast with the liver enzyme, which shows sigmoidal kinetics toward S-adenosylmethionine at all pH values tested [Ogawa and Fujioka (1982) J. Biol. Chem. 257, 3447-3452], the recombinant enzyme exhibited hyperbolic kinetics at low pH and sigmoidal rate behaviour at high pH. The Hill coefficient increased with increasing pH and a pKa of 8.11 was obtained in this transition. The values of Vmax and Km for glycine were not different between the two enzymes. These results suggest that elimination of the positive charge at the N-terminal end either by acetylation or deprotonation is required for co-operative behaviour.
Ogawa
H
H
Department of Biochemistry, Faculty of Medicine Toyama Medical and Pharmaceutical University, Sugitani, Toyama 930-01, Japan.
Gomi
T
T
Takata
Y
Y
Date
T
T
Fujioka
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli; D050938:Glycine N-Methyltransferase; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008780:Methyltransferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism*; D014357:Trypsin
D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; D008780:Methyltransferases; D050938:Glycine N-Methyltransferase; D014357:Trypsin