Glucose modulation of glucokinase activation by small molecules.

Small molecule activators of glucokinase (GK) were used in kinetic and equilibrium binding studies to probe the biochemical basis for their allosteric effects. These small molecules decreased the glucose K0.5 (∼1 mM vs ∼8 mM) and the glucose cooperativity (Hill coefficient of 1.2 vs 1.7) and lowered the kcat to various degrees (62–95% of the control activity). These activators relieved GK’s inhibition from glucokinase regulatory protein (GKRP) in a glucose-dependent manner and activated GK to the same extent as control reactions in the absence of GKRP. In equilibrium binding studies, the intrinsic glucose affinity to the activator-bound enzyme was determined and demonstrated a 700-fold increase relative to the apoenzyme. This is consistent with a reduction in apparent glucose KD and the steady-state parameter K0.5 as a result of enzyme equilibrium shifting to the activator-bound form. The binding of small molecules to GK was dependent on glucose, consistent with the structural evidence for an allosteric b...