Formyltetrahydrofolate Synthetase: BINDING OF ADENOSINE TRIPHOSPHATE AND RELATED LIGANDS DETERMINED BY PARTITION EQUILIBRIUM

Abstract

Abstract The binding of adenine nucleotides and other compounds of related structures to clostridial formyltetrahydrofolate synthetase was examined through the use of the method of partition equilibrium, which utilizes an aqueous biphasic dextran-polyethylene glycol polymer system. The method is rapid and requires minimal amounts of enzyme and ligand. The results of MgATP binding as determined by partition equilibrium agreed well with those obtained by equilibrium dialysis. The enzyme, which has been shown to be tetrameric in structure, possesses four identical, noninteracting nucleotide binding sites per mole of enzyme. MgATP and MgADP bind to the same set of sites. MgATP binding is independent of the presence of the other substrates. The absence of magnesium does not significantly alter the binding of either nucleotide; however, ATP is a less specific ligand than MgATP and is bound to additional sites with a lower affinity. The affinities of other compounds related in structure to ATP were measured by a competitive binding technique. The results of these experiments suggest that the nucleotide site is composed of subsites each of which has a specific binding interaction for a portion of the ATP molecule. Inorganic phosphate can displace MgATP from the enzyme, but it does not affect MgADP binding. The enzyme has a very strong affinity for adenylyl methylenediphosphonate, the β-γ-methylene analogue of ATP, but not for the α-β-methylene analogues of ATP or ADP. Consideration of differences in bond lengths and bond angles between ATP and adenylyl methylenediphosphonate and the specific effect of phosphate on ATP binding suggest that ATP is bound to the enzyme in such a way that the γ-phosphate is strained towards a trigonal-bipyramidal intermediate.