5-Formyltetrahydrofolate is a compound that is administered as a rescue agent in methotrexate chemotherapy and in 5-fluorouracil chemotherapy for synergistic effects. It has also recently been suggested to play a role in bacterial resistance to antifolate therapy. 5,10-methenyltetrahydrofolate synthetase (MTHFS) is the only enzyme known to catalyze the conversion of this compound to 5,10-methenyltetrahydrofolate along with the hydrolysis of ATP to ADP. To better understand the roles of specific amino acids in the ATP binding pocket of this enzyme, we used site-directed mutagenesis to create 10 modified forms of the Mycoplasma pneumoniae ortholog. The Michaelis constant (K(m)) for each substrate and the turnover number (k(cat)) was determined for each mutant to help elucidate the role of individual amino acids. Data were compared to crystal structures of human and M. pneumoniae orthologs of MTHFS. Results were largely consistent with a simple coulombic and proximity model; the larger the predicted charges of an interaction and the closer those interactions were to the phosphate transferred between the substrates, the greater the reduction in ATP binding and catalytic activity of the enzyme.