Mechanisms for auto-inhibition and forced product release in glycine N-methyltransferase: Crystal structures of wild-type, mutant R175K and S-adenosylhomocysteine-bound R175K enzymes

Glycine N-methyltransferase (S-adenosyl-L-methionine: glycine methyltransfe rase, EC 2.1.1.20; GNMT) catalyzes the AdoMet-dependent methylation of glyc ine to form sarcosine (N-methylglycine). Unlike most methyltransferases, GN MT is a tetrameric protein showing a positive cooperativity in AdoMet bindi ng and weak inhibition by S-adenosylhomocysteine (AdoHcy). The first crysta l structure of GNMT complexed with AdoMet showed a unique "closed" molecula r basket structure, in which the N-terminal section penetrates and corks th e entrance of the adjacent subunit. Thus, the apparent entrance or exit of the active site is not recognizable in the subunit structure, suggesting th at the enzyme must possess a second, enzymatically active, "open" structura l conformation. A new crystalline form of the R175K enzyme has been grown i n the presence of an excess of AdoHcy, and its cystal structure has been de termined at 3.0 Angstrom resolution. In this structure, the N-terminal doma in (40 amino acid residues) of each subunit has moved out of the active sit e of the adjacent subunit, and the entrances of the active sites are now op ened widely. An AdoHcy molecule has entered the site occupied in the "close d" structure by Glu15 and Gly16 of the N-terminal domain of the adjacent su bunit. An AdoHcy binds to the consensus AdoMet binding site observed in the other methyltransferase. This AdoHcy binding site supports the glycine bin ding site (Arg175) deduced from a chemical modification study and site-dire cted mutagenesis (R175K). The crystal structures of WT and R175K enzymes we re also determined at 2.5 Angstrom resolution. These enzyme structures have a closed molecular basket structure and are isomorphous to the previously determined AdoMet-GNMT structure. By comparing the open structure to the cl osed structure, mechanisms for auto-inhibition and for the forced release o f the product AdoHcy have been revealed in the GNMT structure. The N-termin al section of the adjacent subunit occupies the AdoMet- binding site and th us inhibits the methyltransfer reaction, whereas the same N-terminal sectio n forces the departure of the potentially potent inhibitor AdoHcy from the active site and thus facilitates the methyltransfer reaction. Consequently GNMT is less active at a low level of AdoMet concentration, and is only wea kly inhibited by AdoHcy. These properties of GNMT are particularly suited f or regulation of the cellular AdoMet/AdoHcy ratio. (C) 2000 Academic Press.