Investigations of Amino Acids in the ATP Binding Site of 5,10-Methenyltetrahydrofolate Synthetase

Abstract

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.

Investigating the Determinants of Substrate Binding through a Semester-Long, Project-Oriented Biochemistry Laboratory Course

“…The most common errors of this type are failure of the site-directed mutagenesis reactions to produce products in the second laboratory period (discovered in the third) and failure to change the codon of the wild-type MTHFS in the second laboratory period (discovered in the fifth). Students encountering these or other catastrophic results are provided with an archived sample from a previous class, a sample from a fellow student, or an archived research , sample. Positive and negative controls are run throughout the course, and these are used to inform technique penalties in grading when failures occur.…”