Convergent Mechanistic Features between the Structurally Diverse N- and O-Methyltransferases: Glycine N-Methyltransferase and Catechol O-Methyltransferase

Abstract

Although an enormous and still growing number of biologically diverse methyltransferases have been reported and identified, a comprehensive understanding of the enzymatic methyl transfer mechanism is still lacking. Glycine N-methyltransferase (GNMT), a member of the family that acts on small metabolites as the substrate, catalyzes methyl transfer from S-adenosyl-l-methionine (AdoMet) to glycine to form S-adenosyl-l-homocysteine and sarcosine. We report primary carbon ((12)C/(14)C) and secondary ((1)H3/(3)H3) kinetic isotope effects at the transferred methyl group, together with (1)H3/(3)H3 binding isotope effects for wild-type GNMT and a series of Tyr21 mutants. The data implicate a compaction effect in the methyl transfer step that is conferred by the protein structure. Furthermore, a remarkable similarity of properties is observed between GNMT and catechol O-methyltransferase, despite significant differences between these enzymes with regard to their active site structures and catalyzed reactions. We attribute these results to a catalytically relevant reduction in the methyl donor-acceptor distance that is dependent on a tyrosine side chain positioned behind the methyl-bearing sulfur of AdoMet.

Scheme 1

Figure 1

Active site of rat GNMT (PDB 1NBH).

Figure 2

Relationship between the k_cat/K_m for glycine and secondary KIE for methylation of glycine by the recombinant rat GNMT and Y21 mutants. r² = 0.93 except for the Y21A mutants. The observation that Y21A is an outliner may indicate a change in active site hydration for this variant (cf. ref (49)).

Figure 3

CH···O hydrogen bond (dashed lines) in the (a) binary GNMT–AdoMet complex (PDB 1NBI) and (b) ternary GNMT–AdoMet–acetate complex (PDB 1NBH).

Scheme 2