Mutational analysis of allosteric activation and inhibition of glucokinase

Abstract

Glucokinase (GK) is activated by glucose binding to the substrate site, is inhibited by GK regulatory protein (GKRP) but stimulated by GK activator drugs (GKAs). To further explore the mechanisms of these processes we studied pure recombinant human GK (normal enzyme and a selection of 31 mutants) using steady state kinetics of the enzyme and tryptophan fluorescence (TF). TF studies of the normal binary GK/glucose complex corroborate recent crystallography showing that it exists in a closed conformation greatly different from the open conformation of the ligand free structure but indistinguishable from the ternary GK/glucose/GKA complex. GKAs did activate and GKRP did inhibit normal GK while its TF was doubled by glucose saturation. However, the enzyme kinetics, GKRP inhibition, TF enhancement by glucose and responsiveness to GKA of the selected mutants varied greatly. Two predominant response patterns were identified accounting for nearly all mutants: 1) GK mutants with a normal or close to normal response to GKA, normally low basal TF (indicating an open conformation), some variability of kinetic parameters (kcat, glucose S0.5, nH and ATP Km) but usually strong GKRP inhibition (13/31); and 2) GK mutants that are refractory to GKAs, exhibit relatively high basal TF (indicating structural compaction and partial closure), usually show strongly enhanced catalytic activity primarily due to lowering of the glucose S0.5 but with reduced or no GKRP inhibition in cases (14/31). These and pertinent literature data are best explained by envisioning a common allosteric regulator region with spatially non overlapping GKRP and GKA binding sites.

A phospho-BAD BH3 helix activates glucokinase by a mechanism distinct from that of allosteric activators

“…A similar series of studies using the M298K mutant in the presence of an allosteric GKA treatment reported V max increase from approximately 60% to 79% of wild-type 34 . However, the restoration of V max in the presence of BAD SAHB A (S118D) occurred with modest effects on S 0.5 of this mutant, which changed from approximately 11 mM to near normal values of 8 mM (Table 2), considerably different from drastic lowering of S 0.5 to approximately 1 mM when M298K is treated with an allosteric GKA 34 . These findings reinforce the mechanistic distinction between allosteric GKAs and SAHB A (S118D) (Table 1).…”

“…Both mutations render the enzyme highly unstable 8,39,40 . Previous reports on the M298K mutation have noted substantial deficiencies in multiple enzymatic parameters 34,36,39,41 . In agreement with these reports, we observed impaired kinetic constants for this mutant, including decreased V max , diminished glucose affinity, and a decreased Hill coefficient (Fig.…”

“…1). These data predict that the binding site for phospho-BAD BH3 helix on GK is distinct from the allosteric site, which becomes available only in the presence of glucose 5,10,12,32,34 .…”

“…GK activators can be expected to promote the protein stability of GK mutants in addition to increasing their activity 34 . We selected two naturally-occurring MODY2 mutations located near the active site, M298K and E300K, for analysis 35–39 .…”

Thermal stabilty of glucokinase (GK) as influenced by the substrate glucose, an allosteric glucokinase activator drug (GKA) and the osmolytes glycerol and urea

“…GK regulatory protein) is a hepato-specific nuclear protein not found in pancreatic islet cells [8, 62]. Also highly relevant for this discussion is the fact that occupancy of the modifier region by GKA and GKRP is mutually exclusive and that glucose facilitates GKA but inhibits GKRP binding and action [4, 65, 67]. The restructuring of the allosteric modifier region and the closure of the substrate site by glucose result in a more compact, more stable enzyme as demonstrated by TF and DSC.…”