Premium
Investigations of the determinants of 5‐formyltetrahydrofolate binding in the active site of 5,10‐methenyltetrahydrofolate synthetase from Mycoplasma pneumoniae
Author(s) -
Johann Timothy,
Wojtera Casey,
Bryant Matthew
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.790.12
Subject(s) - active site , enzyme kinetics , michaelis–menten kinetics , enzyme , chemistry , mutant , circular dichroism , turnover number , site directed mutagenesis , biochemistry , tyrosine , cofactor , stereochemistry , enzyme assay , gene
The pathways for the synthesis of tetrahydrofolates form the core of one carbon metabolism and are an excellent source of targets in the treatment of cancer. 5‐formyltetrahydrofolate (5‐formylTHF) is administered in these treatments, either as a rescue agent or as an enhancer. The only enzyme known to use 5‐formylTHF as a substrate is 5,10‐methenyltetrahydrofolate synthetase (MTHFS). This enzyme catalyzes the conversion of 5‐formylTHF to 5,10‐ methenyltetrahydrofolate in conjunction with the hydrolysis of ATP to ADP. We have investigated the determinants of 5‐ formylTHF binding in the active site of the enzyme through site‐directed mutagenesis of wild type residues to alanine followed by a circular dichroism spectroscopy and kinetics. Replacement of glutamate at position 55 (E55A) or tyrosine at position 123 (Y123A) produced mutants with no activity. Mutant proteins F118A and Y122A demonstrated higher Michaelis constants for 5‐ formylTHF and lower turnover numbers compared to wild type. Curiously, mutant Q144A was found to have a higher Michaelis constant for 5‐formylTHF and a higher turnover number. Circular dichroism data were consistent with all mutants being folded.