Thermodynamics of binding of divalent magnesium and manganese to uridine phosphates: implications for diabetes-related hypomagnesaemia and carbohydrate biocatalysis

Using isothermal titration calorimetry we have studied the binding relationship between the divalent metals, magnesium and manganese, and uridine 5'-phosphates to determine the role these metals play in carbohydrate biosynthesis. It was determined from the isothermal titration calorimetry (ITC) data that Mg+2 and Mn+2 are most tightly bound to PPi, Kb = 41,000 ± 2000 M-1 and 28,000 ± 50,000 M-1 respectively, and UTP, Kb = 14,300 ± 700 M-1 and 13,000 ± 2,000 M-1 respectively.Our results indicate that the formal charge state of the phosphate containing substrates determine the binding strength. Divalent metal cations magnesium and manganese showed similar trends in binding to the sugar substrates. Enthalpy of binding values were all determined to be endothermic except for the PPi case. In addition, entropy of binding values were all found to be positive. From this data, we discuss the role of magnesium and manganese in both sugar nucleotidyltransferase and glycosyltransferase reactions, the differences in metal-bound substrates expected under normal physiological metal concentrations and those of hypomagnesaemia, and the implications for drug design.The roles that nucleotides play in cellular metabolism range in scope from their use as enzyme substrates to regulators for numerous biochemical pathways. Because of their biological importance and the requirement of divalent metals for biological activity, the binding relationship between various metals and nucleotides has been well documented to shed light on the role of metal-nucleotide complexes in the conformational changes that occur in DNA biosynthesis [1-4] and the energy pathways that involve ATP [5-12].Recent studies suggest that certain metabolic disorders are linked to serum magnesium concentrations in a way that can be partially explained by these relative metal-binding equilibria. For example, individuals with hypomagnesaemia – reduced serum magnesium concentration – have an increased incidence of metabolic d