The interaction of the substitution-inert, α, β, γ tridentate complex chromium ATP (CrATP) with 5-phosphoribosyl α-1-pyrophosphate (PP-Rib-P) synthetase from Salmonella typhimurium has been studied by measuring the effects of CrATP on the kinetics of the PP-Rib-P synthesis reaction and on the nuclear magnetic relaxation rates of the anomeric protons and phosphorus atom of ribose 5-phosphate, to determine the intersubstrate distances on the enzyme. CrATP is a linear competitive inhibitor with respect to MgATP and a linear uncompetitive inhibitor with respect to ribose 5-phosphate, with similar K i values (0.30 ± 0.02 n m) indicating that CrATP occupies the MgATP site and that ribose 5-phosphate is also bound. From the paramagnetic effects of enzyme-bound CrATP on the longitudinal relaxation rates of the anomeric protons and phosphorus atom of ribose 5-phosphate in the enzyme-CrATP-ribose-5-phosphate complex, three distances from Cr 3+ to ribose 5-phosphate are obtained, ranging from 6.7 to 8.0 Å. The data and a model of the bound substrates based on the absolute and relative distances indicate that both the α and β anomers of ribose 5-phosphate bind to the enzyme near the bound CrATP, and that the H-1 of the β anomer is ~1.0 Å closer to bound CrATP than is the H-1 of the α anomer. Hence the 1-OH group of the α anomer is ~1.6 Å closer to the bound CrATP than the 1-OH group of the β anomer. From this model, the lower limit distance from the 1-OH of α-ribose 5-phosphate to the β-P of CrATP on the enzyme approximates van der Waals contact, which is consistent with a direct S N2 nucleophilic displacement of AMP from ATP by α-ribose 5-phosphate. Moreover, the nmr data indicate that the β-anomer of ribose 5-phosphate is incorrectly oriented for nucleophilic attack on CrATP. The nmr data also establish the close proximity of a bound inorganic phosphate ion 7.1 ± 0.7 Å from the binding site for CrATP, which may represent the phosphate ion required for enzyme activity.