Residues involved in the mechanism of the bifunctional methylenetetrahydrofolate dehydrogenasecyclohydrolase

The human bifunctional dehydrogenase/cyclohydrolase domain catalyzes the interconversion of 5,10-methyleneH4folate and 10-formylH 4folate. Although previous structure and mutagenesis studies indicated the importance of lysine56 in cyclohydrolase catalysis, the role of several surrounding residues had not been explored. In addition to further defining the role of lysine56, the work presented in this thesis explores the functions of glutamine100 and aspartate125 through the use of site-directed mutagenesis and chemical modification. The results indicate that the function of glutamine100 is to polarize lysine56 for cyclohydrolase catalysis, and that of aspartate125 is for the binding of the H4folate substrates. In highlighting the importance of these residues, a new catalytic mechanisms is proposed for both activities, as well as a novel explanation for the differences in channeling between the forward and reverse directions