ATP and ADP bind to N10-CHO-H4folate synthetase from Clostridium cylindrosporum at four identical sites. Although both ADP and ATP bind to the enzyme with essentially the same Ka values as the Mg2+.nucleotide complexes, only the Mg2+.nucleotides are kinetically active. Using transferred nuclear Overhauser effect (TRNOE) NMR spectroscopy, we have measured the time-dependent NOE buildup rates of selected protons in ADP and ATP bound to N10-CHO-H4folate synthetase after preirradiating protons H1', H2', H3', and H4'. The results were used to calculate interproton distances. In order to define the conformations of ADP and ATP bound to the enzyme, we used the TRNOE distance constraints in a distance geometry algorithm. The results of the distance geometry calculations suggest that, within experimental error, the conformations of both ADP and ATP (with or without Mg2+) have an average glycosidic torsion angle X (O4'-C1'-N9-C8) of 100 degrees +/- 20 degrees and a sugar pucker angle psi' (C5'-C4'-C3'-O3') of 85 degrees +/- 5 degrees. These values are consistent with a nucleotide structure generated by computer modeling after energy minimization, which has X = 90 degrees +/- 6 degrees and psi' = 81 degrees, indicating a high-anti and C3'-endo conformation.