title: Specific potassium ion interactions facilitate homocysteine binding to betaine-homocysteine S-methyltransferase.
abstract: 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 K(M) for K⁺ of about 100 µM. The presence of potassium ions lowers the apparent K(M) of the enzyme for homocysteine, but it does not affect the apparent K(M) for betaine or the apparent k(cat) 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.
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., Flemingovo nám. 2, 166 10 Prague 6, Czech Republic.
authors.forename: Jana
authors.initials: J
authors.lastname: Mládková
authors.forename: Jana
authors.initials: J
authors.lastname: Hladílková
authors.forename: Carrie E
authors.initials: CE
authors.lastname: Diamond
authors.forename: Katherine
authors.initials: K
authors.lastname: Tryon
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Yamada
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Garrow
authors.forename: Pavel
authors.initials: P
authors.lastname: Jungwirth
authors.forename: Markos
authors.initials: M
authors.lastname: Koutmos
authors.forename: Jiří
authors.initials: J
authors.lastname: Jiráček
mesh_terms: D001622:Betaine / Q000737:chemistry / Q000378:metabolism; D050940:Betaine-Homocysteine S-Methyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D030562:Databases, Protein; D004789:Enzyme Activation; D006710:Homocysteine / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011188:Potassium / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; D006710:Homocysteine; D001622:Betaine; C494548:BHMT protein, human; D050940:Betaine-Homocysteine S-Methyltransferase; D011188:Potassium
keywords: BHMT; crystal structure; enzyme kinetics; homocysteine; molecular dynamics; potassium; simulations