Metal ion and substrate binding to bovine galactosyltransferase.

Bovine milk galactosyltransferase was examined by ESR and NMR proton relaxation measurements to determine the stoichiometry and nature of manganese and UDP-Gal substrate binding. The ESR and NMR data clearly showed the binding of two (Mn(II) per mol of enzyme in the ternary complex (enzyme-manganese-UDP-Gal). The affinity of the enzyme for manganese is much higher in the presence of UDP-Gal than in its absence. A deenhancement was observed in both water and UDP-Gal proton relaxation rates upon ternary complex formation [enzyme-Mn(II)-UDP-Gal] relative to the metal-substrate [Mn(II)-UDP-Gal] binary complex, yet the temperature dependence of the water proton relaxation rate was consistent with fast exchange. A simple model was proposed which accounted for the pronounced deenhancement, involving a slow conformational interconversion of an initially formed, rapidly exchanging conformer of the enzyme-Mn(II)-UDP-Gal complex to a second form which contributes negligibly to the relaxation.