Dissecting the nucleotide binding properties of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase with fluorescent 3 '(2)'-o-anthraniloyladenosine 5 '-triphosphate

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the tr ansfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin, the first reaction in the folate biosynthetic pathway. Like other enzymes in th e folate pathway, HPPK is an ideal target for development of antimicrobial agents because the enzyme is essential for microorganisms but is absent fro m humans and animals. Using 3'(2')-o-anthraniloyladenosine 5'-triphosphate as a fluorescent probe, a fluorometric competitive binding assay has been d eveloped for measuring the dissociation constants of various compounds that bind to the ATP site of HPPK. The fluorometric assay has been used to dete rmine the nucleotide specificity and dissect the energetics of the binding of MgATP. The order of affinity of various nucleoside triphosphates for HPP K is MgATP > MgGTP > MgITP > MgXTP approximate to MgUTP approximate to MgCT P. The affinity of MgATP for HPPK (K-d = 2.6 +/- 0.06 mu M) is 260-fold hig her than that of MgGTP and more than 1000-fold higher than those of the oth er nucleoside triphosphates, indicating that HPPK is highly specific with r espect to the base moiety of the nucleotide. The affinity of ATP for HPPK i n the presence of Mg2+ is 15 times that in the absence of Mg2+, 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 similar to 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 amon g the three phosphoryl groups of MgATP, the gamma-phosphoryl group is most critical for binding to HPPK and the alpha-phosphoryl group contributes lit tle to the binding of the nucleotide. The affinity of MgATP is 18 times tha t of MgdATP, indicating that the 2'-hydroxyl group of MgATP is also importa nt for binding. van't Hoff analysis suggests that binding of MgATP is mainl y driven by enthalpy at 25 degrees C and the entropy of binding is also in favor of the formation of the HPPK MgATP complex. (C) 2000 Elsevier Science B.V. All rights reserved.