Betaine-homocysteine S-methyltransferase (BHMT) is a zinc-dependent methyltransferase that uses 
betaine as the methyl donor for the remethylation of homocysteine to form methionine. This 
reaction supports S-adenosylmethionine biosynthesis, which is required for hundreds of methylation
reactions in humans. Herein we report that BHMT is activated by potassium ions with an apparent 
KM for K+ of about 100 .M. The presence of potassium ions lowers the apparent KM of the enzyme 
for homocysteine, but it does not affect the apparent KM for betaine or the apparent kcat for 
either substrate. We employed molecular dynamics (MD) simulations to theoretically predict and 
protein crystallography to experimentally localize the binding site(s) for potassium ion(s). 
Simulations predicted that K+ ion would interact with residues Asp26 and/or Glu159. Our crystal 
structure of BHMT bound to homocysteine confirms these sites of interaction and reveals further 
contacts between K+ ion and BHMT residues Gly27, Gln72, Gln247 and Gly298. The potassium binding 
residues in BHMT partially overlap with the previously identified DGG (Asp26-Gly27-Gly28) 
fingerprint in the Pfam 02574 group of methyltransferases. Subsequent biochemical characterization
of several site-specific BHMT mutants confirmed the results obtained by the MD simulations and 
crystallographic data. Together, the data herein indicate that the role of potassium ions in BHMT 
is structural and that potassium ion facilitates the specific binding of homocysteine to the 
active site of the enzyme.