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 (PP_in) inhibits or activates glycerol kinase due to the complexation of PP_in 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.

Cellulomonas sp。的甘油激酶的详细动力学和机理分析。关于Mg ²⁺与ATP的摩尔比进行。该酶基本上需要Mg ²⁺才能发挥其活性，并且在最佳Mg ²⁺与ATP摩尔比为[0.12-0.3]时显示出最大的活性。随后，Mg ²⁺与ATP摩尔比的增加高于最佳区域中的值，则将酶活性抑制为一个非零的渐近值。由于在不同的Mg ²⁺与ATP摩尔比下形成了多个Mg-ATP络合物，因此在固定的Mg ²⁺浓度下，该酶表现出两步动力学作为ATP的函数。无机多磷酸盐（PP _in）由于PP _i n与Mg ²⁺的络合而抑制或激活了甘油激酶，Mg ²⁺使Mg ²⁺与ATP的摩尔比降低至或低于最佳水平。通过添加Mg ²⁺改变ATP的所有³¹ P NMR信号（即α-，β-和γ-磷酸盐）的变化表明，它们全部都与Mg-ATP络合物的形成有关。加入Mg ²⁺确立了最佳Mg ²⁺的上限后，ATP核糖部分上CH ₂质子的¹ H NMR信号几乎相等。与ATP的摩尔比表示酶的活性。因此，可以得出结论，相对于不同的Mg-ATP复合物，甘油激酶的活性位点显示出不同的催化性能。甘油激酶对具有化学计量或化学计量过量组成的复合物（例如Mg ₂ ATP ）表现出高亲和力（低 K _m）和较低活性（低k _cat）。另一方面，该酶对[Mg（ATP）₂ ] ^-6等不饱和复合物显示出较低的亲和力（较高的 K _m）和较高的活性（较高的k _cat）。