The interactions of mandelate racemase with divalent metal ion, substrate, and competitive inhibitors were investigated. The enzyme was found by electron paramagnetic resonance (EPR) to bind 0.9 Mn2+ ion per subunit with a dissociation constant of 8 muM, in agreement with its kinetically determined activator constant. Also, six additional Mn2+ ions were found to bind to the enzyme, much more weakly, with a dissociation constant of 1.5 mM. Binding to the enzyme at the tight site enhances the effect of Mn2+ on the longitudinal relaxation rate (1/T1p) of water protons by a factor of 11.9 at 24.3 MHz. From the frequency dependence of 1/T1p, it was determined that there are similar to 3 water ligands on enzyme-bound Mn2+ which exchange at a rate larger than or equal to 10-7 sec-1. The correlation time for enzyme-bound Mn2+-water interaction is frequency-dependent, indicating it to be dominated by the electron spin relaxation time of Mn2+. Formation of the ternary enzyme-Mn2+-mandelate complex decreases the number of fast exchanging water ligands by similar to 1, but does not affect tau-c, suggesting the displacement or occlusion of a water ligand. The competitive inhibitors D,L-alpha-phenylglycerate and salicylate produce little or no change in the enzyme-Mn2+-H2O interaction, but ternary complexes are detected indirectly by changes in the dissociation constant of the enzyme-Mn2+ complex and by mutual competition experiments. In all cases the dissociation constants of substrates and competitive inhibitors from ternary complexes determined by magnetic resonance titrations agree with K-M and K-i values determined kinetically and therefore reflect kinetically active complexes. From the paramagnetic effects of Mn2+ on 1/T1 and 1/T2 of the 13C-enriched carbons of 1-[13C]-D,L-mandelate and 2-[13C]-D,L-mandelate, Mn2+ to carboxylate carbon and Mn2+ to carbinol carbon distances of 2.93 plus or minus 0.04 and 2.71 plus or minus 0.04 A, respectively, were calculated, indicating bidentate chelation in the binary Mn2+-mandelate complex. In the active ternary complex of enzyme, Mn2+, and D,L-mandelate, these distances increase to 5.5 plus or minus 0.2 and 7.2 plus or minus 0.2 A, respectively, indicating the presence of at least 98.9% of a second sphere complex in which Mn2+, and C1 and C2 carbon atoms are in a linear array. The water relaxation data suggest that a water ligand is immobilized between the enzyme-bound Mn2+ and the carboxylate of the bound substrate. This intervening water ligand may polarize or protonate the carboxyl group. From 1/T2p the rate of dissociation of the substrate from this ternary complex (larger than or equal to 5.6 times 10-4 sec-1) is at least 52 times greater than the maximal turnover number of the enzyme (1070 sec-1), indicating that the complex detected by nuclear magnetic resonance (NMR) is kinetically competent to participate in catalysis. Relationships among the microscopic rate constants are considered.