The Protein Journal

, 27:303

Investigations of the Roles of Arginine 115 and Lysine 120 in the Active Site of 5,10-Methenyltetrahydrofolate Synthetase from Mycoplasma pneumoniae

  • Amber N. Hancock
  • R. Shane Coleman
  • Richard T. Johnson
  • Catherine A. Sarisky
  • Timothy W. Johann
Article

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 (Km) for ATP from 76 to 1,200 μM and the Km for 5-formylTHF from 2.5 to 7.1 μM. 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.

Keywords

5-Formyltetrahydrofolate Site-directed mutagenesis 5,10-Methenyltetrahydrofolate synthetase ATP Kinetics 

Abbreviations

5-formylTHF

5-Formyltetrahydrofolate

5,10-methenylTHF

5,10-Methenyltetrahydrofolate

ADP

Adenosine diphosphate

ATP

Adenosine triphosphate

CD

Circular dichroism spectroscopy

HEPES

N-Cyclohexyl-2-aminoethanesulfonic acid

Km

Michaelis constant

LB

Luria-Bertani

MES

2-(N-Morpholino)ethanesulfonic acid

MTHFS

5,10-Methenyltetrahydrofolate synthetase

PCR

Polymerase chain reaction

PBS

Phosphate buffered saline

THF

Tetrahydrofolate

UV

Ultraviolet

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Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Amber N. Hancock
    • 1
  • R. Shane Coleman
    • 2
  • Richard T. Johnson
    • 2
  • Catherine A. Sarisky
    • 3
  • Timothy W. Johann
    • 2
  1. 1.Department of ChemistryVirginia TechBlacksburgUSA
  2. 2.Department of Chemistry and PhysicsRadford UniversityRadfordUSA
  3. 3.Department of ChemistryRoanoke CollegeSalemUSA

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