A new, more gentle enzyme purification for yeast enolase was developed. A series of kinetic experiments was performed with yeast enolase where the concentration of Mg(II) is kept constant and at the K(m)' level; the addition of Mn(II), Zn(II), or Cu(II) gives a hyperbolic decrease in the enzyme activity. The final velocity of these mixed-metal systems is the same as the velocity obtained only with Mn(II), Zn(II), or Cu(II), respectively. The concentration of the second metal that gives half-maximal effect in the presence of Mg(II) is approximately the same as the apparent K(m) (K(m)') value measured for that cation alone. Direct binding of Mn(II) to apoenolase in the absence and presence of Mg(II) shows that Mn(II) and Mg(II) compete for the same metal site on enolase. In the presence Of D-2-phosphoglycerate (PGA) and Mg(II), only a single cation site per monomer is occupied by Mn(II). Water proton relaxation rate (PRR) studies of enzyme-ligand complexes containing Mn(II) and Mn(II) in the presence of Mg(II) are consistent with Mn(II) binding at site I under both conditions. PRR titrations of ligands such as the substrate PGA or the inhibitors orthophosphate or fluoride to the enolase-Mn(II)-Mg(II) complex are similar to those obtained for the enolase-Mn(II) complex, also indicating that Mn(II) is at site I in the presence of Mg(II). High-resolution H-1 and P-31 NMR was used to determine the paramagnetic effect of enolase-bound Mn(II) on the relaxation rates of the nuclei of the competitive inhibitor phosphoglycolate. The distances between the bound Mn(II) and the nuclei were calculated. In the presence of Mg(II) at site II, the distance between Mn(II) and the protons of phosphoglycolate was 5.73 angstrom and the distance between Mn(II) and the phosphorus of phosphoglycolate was 5.28 angstrom. In the absence of Mg(II), these distances were 6.00 and 6.59 angstrom, respectively. These results indicate that the presence of Mg(II) at site II causes an effect on the structure of the ligand at the active site. The frequency dependence of the PRR of various enolase-metal complexes was measured. The enolase-Mn-PGA, enolase-Mn-PGA-Mg, and the enolase-Mn-PGA-Mn complexes give similar frequency dispersions. The tau(c) values for these complexes calculated for 24.3 MHz were 6.65, 4.31, and 6.24 ns, respectively. The number of exchangeable water molecules coordinated to the bound Mn(II) was estimated to be 0.5 for each of these complexes. The binding of Mg(II) or Mn(II) at site II appears not to affect the properties of Mn(II) at site I, suggesting that the metal sites are far apart in the enzyme complex and are magnetically independent. The tau(c) and the number of water molecules were also calculated from a measurement of the T1p/T2p ratio of water at 300 MHz. The second metal ion appears to be > 12 angstrom from the metal site I and not at the catalytic site or involved in the catalytic process. The cation at site I influences the catalytic constant of the enzyme.
