6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7, 8-dihydropterin, the first reaction in the folate biosynthetic pathway. Like other enzymes in the folate pathway, HPPK is an ideal target for development of antimicrobial agents because the enzyme is essential for microorganisms but is absent from humans and animals. Using 3'(2')-o-anthraniloyladenosine 5'-triphosphate as a fluorescent probe, a fluorometric competitive binding assay has been developed for measuring the dissociation constants of various compounds that bind to the ATP site of HPPK. The fluorometric assay has been used to determine the nucleotide specificity and dissect the energetics of the binding of MgATP. The order of affinity of various nucleoside triphosphates for HPPK is MgATP>MgGTP>MgITP>MgXTP approximately MgUTP approximately MgCTP. The affinity of MgATP for HPPK (K(d)=2.6+/-0.06 microM) is 260-fold higher than that of MgGTP and more than 1000-fold higher than those of the other nucleoside triphosphates, indicating that HPPK is highly specific with respect to the base moiety of the nucleotide. The affinity of ATP for HPPK in the presence of Mg(2+) is 15 times that in the absence of Mg(2+), indicating that the metal ion is important for the binding of the nucleotide. Removal of the gamma-phosphate from MgATP reduces its affinity for HPPK by a factor of approximately 21. The affinity of AMP for HPPK is about one third that of ADP and almost the same as that of adenosine. The result suggests that among the three phosphoryl groups of MgATP, the gamma-phosphoryl group is most critical for binding to HPPK and the alpha-phosphoryl group contributes little to the binding of the nucleotide. The affinity of MgATP is 18 times that of MgdATP, indicating that the 2'-hydroxyl group of MgATP is also important for binding. van't Hoff analysis suggests that binding of MgATP is mainly driven by enthalpy at 25 degrees C and the entropy of binding is also in favor of the formation of the HPPK.MgATP complex.