title: Investigations of the roles of arginine 115 and lysine 120 in the active site of 5,10-methenyltetrahydrofolate synthetase from Mycoplasma pneumoniae.
abstract: 5,10-Methenyltetrahydrofolate synthetase (MTHFS) catalyzes the conversion of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate coupled to the hydrolysis of ATP. A co-crystal structure of MTHFS bound to its substrates has been published (Chen et al., Proteins 56:839-843, 2005) that provides insights into the mechanism of this reaction. To further investigate this mechanism, we have replaced the arginine at position 115 and the lysine at position 120 with alanine (R115A and K120A, respectively). Circular dichroism spectra for both mutants are consistent with folded proteins. R115A shows no activity, suggesting that R115 plays a critical role in the activity of the enzyme. The K120A mutation increases the Michaelis constant (K(m)) for ATP from 76 to 1,200 microM and the K(m) for 5-formylTHF from 2.5 to 7.1 microM. The weaker binding of substrates by K120A may be due to movement of a loop consisting of residues 117 though 120, which makes several hydrogen bonds to ATP and may be held in position by K120.
authors.affiliation: Department of Chemistry, Virginia Tech, Blacksburg, VA 24061, USA.
authors.forename: Amber N
authors.initials: AN
authors.lastname: Hancock
authors.forename: R Shane
authors.initials: RS
authors.lastname: Coleman
authors.forename: Richard T
authors.initials: RT
authors.lastname: Johnson
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Sarisky
authors.forename: Timothy W
authors.initials: TW
authors.lastname: Johann
mesh_terms: D001120:Arginine / Q000235:genetics / Q000378:metabolism; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D002942:Circular Dichroism; D004789:Enzyme Activation; D007700:Kinetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D009177:Mycoplasma pneumoniae / Q000201:enzymology* / Q000235:genetics
chemical_list: D001120:Arginine; D019731:Carbon-Nitrogen Ligases; C021557:5,10-methenyltetrahydrofolate synthetase; D008239:Lysine