Recombinant expression of rat glycine N-methyltransferase and evidence for contribution of N-terminal acetylation to co-operative binding of S-adenosylmethionine

Abstract

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 α-amino group of rat glycine N-methyltransferase is blocked by acetylation. In contrast with the liver enzyme, which shows sigmoidal kinetics toward S-adenosylmethionine at all pH values tested, 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 pK(a) of 8.11 was obtained in this transition. The values of V(max) and K(m) 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.