Kinetic Analysis of β-Phosphoglucomutase and Its Inhibition by Magnesium Fluoride

The isomerization of β-glucose-1-phosphate (βG1P) to β-glucose-6-phosphate (G6P) catalyzed by β-phosphoglucomutase (βPGM) has been examined using steady- and presteady-state kinetic analysis. In the presence of low concentrations of β-glucose-1,6-bisphosphate (βG16BP), the reaction proceeds through a Ping Pong Bi Bi mechanism with substrate inhibition (k_cat = 65 s⁻¹, K_βG1P = 15 μM, K_βG16BP = 0.7 μM, K_i = 122 μM). If αG16BP is used as a cofactor, more complex kinetic behavior is observed, but the nonlinear progress curves can be fit to reveal further catalytic parameters (k_cat = 74 s⁻¹, K_βG1P = 15 μM, K_βG16BP = 0.8 μM, K_i = 122 μM, K_αG16BP = 91 μM for productive binding, K_αG16BP = 21 μM for unproductive binding). These data reveal that variations in the substrate structure affect transition-state affinity (approximately 140000-fold in terms of rate acceleration) substantially more than ground-state binding (110-fold in terms of binding affinity). When fluoride and magnesium ions are present, time-dependent inhibition of the βPGM is observed. The concentration dependence of the parameters obtained from fitting these progress curves shows that a βG1P·MgF₃⁻·βPGM inhibitory complex is formed under the reaction conditions. The overall stability constant for this complex is approximately 2 × 10⁻¹⁶ M⁵ and suggests an affinity of the MgF₃⁻ moiety to this transition-state analogue (TSA) of ≤70 nM. The detailed kinetic analysis shows how a special type of TSA that does not exist in solution is assembled in the active site of an enzyme. Further experiments show that under the conditions of previous structural studies, phosphorylated glucose only persists when bound to the enzyme as the TSA. The preference for TSA formation when fluoride is present, and the hydrolysis of substrates when it is not, rules out the formation of a stable pentavalent phosphorane intermediate in the active site of βPGM.