Catalytic Mechanism of Glycine N-Methyltransferase^,

Abstract

Methyltransfer reactions are some of the most important reactions in biological systems. Glycine N-methyltransferase (GNMT) catalyzes the S-adenosyl-l-methionine- (SAM-) dependent methylation of glycine to form sarcosine. Unlike most SAM-dependent methyltransferases, GNMT has a relatively high value and is weakly inhibited by the product S-adenosyl-l-homocysteine (SAH). The major role of GNMT is believed to be the regulation of the cellular SAM/SAH ratio, which is thought to play a key role in SAM-dependent methyltransfer reactions. Crystal structures of GNMT complexed with SAM and acetate (a potent competitive inhibitor of Gly) and the R175K mutated enzyme complexed with SAM were determined at 2.8 and 3.0 A resolutions, respectively. With these crystal structures and the previously determined structures of substrate-free enzyme, a catalytic mechanism has been proposed. Structural changes occur in the transitions from the substrate-free to the binary complex and from the binary to the ternary complex. In the ternary complex stage, an alpha-helix in the N-terminus undergoes a major conformational change. As a result, the bound SAM is firmly connected to protein and a "Gly pocket" is created near the bound SAM. The second substrate Gly binds to Arg175 and is brought into the Gly pocket. Five hydrogen bonds connect the Gly in the proximity of the bound SAM and orient the lone pair orbital on the amino nitrogen (N) of Gly toward the donor methyl group (C(E)) of SAM. Thermal motion of the enzyme leads to a collision of the N and C(E) so that a S(N)2 methyltransfer reaction occurs. The proposed mechanism is supported by mutagenesis studies.

Acetylation of N-terminal valine of glycine N-methyltransferase affects enzyme inhibition by folate

“…While this did not result in a higher binding constant for folate it could result in a stronger interaction of the acetylated valine with the hydrophobic globular part of the protein in the GNMT-folate complex. The mechanism of GNMT action has been proposed to involve the movement of the interacting N-termini to expose entry of AdoMet to the active site [17]. Binding of folate to this region would diminish the flexibility of the N-termini and explain the inhibition.…”

“…GNMT has been isolated and studied from a variety of sources [11][12][13]; inhibition of enzyme activity by folate was described in some detail [14] and several crystal structures were solved [15][16][17][18]. Recently the crystal structure of the GNMT-folate complex was reported [19].…”

Thermodynamic Evaluation of Ligand Binding in the Plant-like Phosphoethanolamine Methyltransferases of the Parasitic Nematode Haemonchus contortus

“…For example, glycine methyltransferase, which is a tetrameric enzyme, undergoes cooperative changes in each monomer upon AdoMet binding (38,39). Likewise, the crystal structures of the guanidoacetate-, phenylethanolamine-, and histamine-N-methyltransferases reveal that different parts of their respective N-terminal regions act as a lid domain that shifts into the active site upon binding of either AdoMet or AdoCys binding (40 -42).…”

Structural and Functional Studies of Pavine N-Methyltransferase from Thalictrum flavum Reveal Novel Insights into Substrate Recognition and Catalytic Mechanism

“…The architectures of the active sites in more distantly related NMTs appear even more divergent than between PfPMT and PavNMT. A variety of catalytic mechanisms have been proposed for rat liver glycine NMT (17), rat liver guanidinoacetate NMT (18), and human phenylethanolamine NMT (19,20). In these other NMTs, a variety of functional groups have been proposed for general base catalysis and for the stabilization of the transition state.…”