Mechanistic and kinetics elucidation of Mg²⁺/ATP molar ratio effect on glycerol kinase

Detailed kinetics and mechanistic analyses of glycerol kinase from Cellulomonas sp. with respect to Mg²⁺ to ATP molar ratio were performed. The enzyme essentially requires Mg²⁺ for its activity and shows maximum activity at the optimum Mg²⁺ to ATP molar ratio of [0.12–0.3]. Subsequent increase of Mg²⁺ to ATP molar ratio higher than the values in the optimum region suppresses the enzyme activity to a non-zero asymptotic value. The enzyme exhibits two-step kinetics as a function of ATP at a fixed Mg²⁺ concentration due to the formation of multiple Mg-ATP complexes at different Mg²⁺ to ATP molar ratio. The addition of inorganic polyphosphate (PPin) inhibits or activates glycerol kinase due to the complexation of PPin with Mg²⁺ that shifts Mg²⁺ to ATP molar ratio below or to the optimum level. The change in all 31P NMR signals of ATP (i.e. α-, β- and γ-phosphate) by the addition of Mg²⁺ reveals that all of them are involved in Mg-ATP complex formation. The 1H NMR signals of CH2-protons on the ribose moiety of ATP become nearly equivalent after the addition of Mg²⁺ establishing the upper limit of optimum Mg²⁺ to ATP molar ratio for the enzyme activity. Therefore, it is concluded that the active site of glycerol kinase shows different catalytic property with respect to different Mg-ATP complexes. Glycerol kinase exhibits high affinity (low K_m) and less activity (low k_cat) for complexes with a stoichiometric or over-stoichiometric constitution like Mg₂ATP. On the other hand, the enzyme shows less affinity (high K_m) and high activity (high k_cat) for unsaturated complexes like [Mg(ATP)2]⁻⁶.