title: Residues involved in the mechanism of the bifunctional methylenetetrahydrofolate dehydrogenase-cyclohydrolase: the roles of glutamine 100 and aspartate 125.
abstract: The human bifunctional dehydrogenase-cyclohydrolase domain catalyzes the interconversion of 5,10-methylene-H(4)folate and 10-formyl-H(4)folate. Although previous structure and mutagenesis studies indicated the importance of lysine 56 in cyclohydrolase catalysis, the role of several surrounding residues had not been explored. In addition to further defining the role of lysine 56, the work presented in this study explores the functions of glutamine 100 and aspartate 125 through the use of site-directed mutagenesis and chemical modification. Mutants at position 100 are inactive with respect to cyclohydrolase activity while preserving significant dehydrogenase levels. We succeeded in producing a K56Q/Q100K double mutant, which has no cyclohydrolase yet retains more than two-thirds of wild type dehydrogenase activity. Neither activity is detectable in aspartate 125 mutants with the exception of D125E. The results indicate that the function of glutamine 100 is to activate lysine 56 for cyclohydrolase catalysis and that aspartate 125 is involved in the binding of the H(4)folate substrates. In highlighting the importance of these residues, catalytic mechanisms are proposed for both activities as well as an explanation for the differences in channeling efficiency in the forward and reverse directions.
authors.affiliation: Department of Biochemistry, McIntyre Medical Sciences Building, McGill University, Montreal, Quebec H3G 1Y6, Canada.
authors.forename: Saravanan
authors.initials: S
authors.lastname: Sundararajan
authors.forename: Robert E
authors.initials: RE
authors.lastname: MacKenzie
mesh_terms: D001224:Aspartic Acid / Q000378:metabolism*; D002384:Catalysis; D005973:Glutamine / Q000378:metabolism*; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation
chemical_list: D005973:Glutamine; D001224:Aspartic Acid; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP)