Bovine Galactosyl Transferase. Substrate . Manganese Complexes and the Role of Manganese Ions in the Mechanism

Abstract

The role of Mn2+ in the reaction catalysed by the galactosyl transferase of bovine milk and colostrum was studied by steady-state kinetic methods at pH 7.4 and 37°C. The association constants for the binding of Mn2+ by UDP-galactose and by the buffer species used (3-[N-morpho-lino]propanesulphonate) were measured by electron spin resonance spectroscopy and the concentrations of all the species present in the substrate mixture of Mn2+· UDP-galactose and N-acetyl-glucosamine were calculated. Previous evidence indicated that high Mn2+ concentrations activate the reaction beyond what is expected from the Michaelis-Menten equation. At such levels (up to 40 mM), the concentration of free Mn2+ was found to be proportional to total Mn within 3%. Experimental data produced as a function of total Mn concentration and total UDP-galactose (i.e. free UDP-galactose plus MnUDP-galactose) concentration could be interpreted in terms of rate equations written in relatively simple form with free Mn2+ and total UDP-galactose concentrations as variables, corresponding to a large variety of kinetic models involving Mn2+, UDP-galactose and MnUDP-galactose as substrates. A group of related mechanisms could not be eliminated at this stage and experiments were carried out in which the concentrations of two of the substrates were varied at either a constant ratio or a constant product depending on how they were involved in the Mn2+: UDP-galactose association reaction. A choice between the two final mechanisms was made by applying statistical non-linear least-squares methods. The most likely mechanism was one in which, after the addition of Mn2+ to the enzyme, the substrates UDP-galactose and MnUDP-galactose added as alternatives to produce two branches, in each of which was added N-acetylglucosamine followed by the release of N-acetyllactosamine, then the Mn2+ complex of UDP, and finally (in the branch in which MnUDP-galactose had been the substrate) free Mn2+.