Given the proposed importance of conformational

Given the proposed importance of conformational dynamics to GCK's allosteric regulation, a number of biophysical methods have been utilized to probe the enzyme's dynamic structural landscape. Investigations into the mechanism of BAY 41-8543 sale binding, using changes in the enzyme's intrinsic fluorescence to report on structure, confirmed that GCK experiences slow dynamics [35]. Two different transient-state stopped-flow studies provided compelling evidence in support of the LIST mechanism by demonstrating the existence of multiple unliganded enzyme conformations [36,37]. Carr-Purcell-Meiboom-Gill (CPMG) NMR experiments provided an estimate for the rate constant of conformational exchange between a putative ground and “excited” conformation of the enzyme, verifying a temporal equivalency between conformational exchange and enzymatic turnover [38]. Pulsed proteolysis studies demonstrated that the flexible active site loop is the primary site of disorder in unliganded GCK, consistent with NMR and crystallographic data indicating the small domain retains a high degree of order in the absence of glucose [38]. NMR studies also showed that glucose binding promotes folding of the flexible active site loop, quenches the millisecond dynamics of the small domain, and substantially narrows the protein's conformational ensemble [39]. Together this information provides a detailed atomistic description of the structural and dynamic origins of GCK's allosteric regulation by glucose. The molecular picture of GCK emerging from the studies summarized above provides a framework for understanding the mechanistic basis of hyperactive, PHHI-associated GCK variants that lack cooperativity. Based on these studies, loss of cooperativity is expected to occur from alterations in the rate constants dictating turnover (kcat) and/or conformational exchange (kex). In line with that expectation, two functionally distinct GCK activation mechanisms were recently identified by characterizing genetically engineered GCK mutants: α- and β-type activation [40]. Specific mutations in GCK's α13 helix give rise to a variant that symbolizes α-type activation. β-type activation, on the other hand, is exemplified by β-hairpin GCK, which contains targeted alterations in the mobile loop region. Although both activation schemes are operational in clinically identified mutants, the α13 helix and β-hairpin GCK variants have shown substantial utility in developing a molecular understanding of each activation process. Solution NMR, viscosity variation, transient chemical quench-flow kinetics and hydrogen-deuterium exchange mass spectrometry (HDX-MS) were recently used to elucidate the mechanism of action of both variants [40]. These combined efforts demonstrate that unliganded α-activated GCK is characterized by a shift in the conformational ensemble toward a structure that resembles the glucose-bound enzyme. Consequently, α-activation is associated with an alteration in the value of the conformational exchange rate constant, kex. This shift is accompanied by an increase in glucose affinity and decreased proteolytic susceptibility within the flexible loop. β-activated GCK, however, appears to be structurally identical to unliganded wild type GCK and activation results from an accelerated product release step. This mutant also displays enhanced proteolytic susceptibility of the active site mobile loop [38], suggesting that swift product release is facilitated by increased dynamics of the mobile loop. Interestingly, GKAs that have been explored as potential MODY therapeutics appear to operate via the α-type activation mechanism [41].
Inhibition of GCK by the glucokinase regulatory protein The earliest identified, and to date best characterized GCK interaction partner is the liver-specific glucokinase regulatory protein (GKRP, GRP). First discovered by VanSchaftingen nearly 30 years ago [42,43], GKRP has been extensively studied using a variety of biochemical, biophysical, structural and cellular methods. Importantly, this chapter discusses hepatic GCK regulation exclusively, as this interaction occurs only in liver hepatocytes and not in pancreatic β-cells. Below we provide a brief summary of the salient features of the GCK-GKRP interaction with a special focus on studies conducted since 2013, when crystal structures of the mammalian and xenopus GCK-GKRP complexes first appeared [44,45]. For a more in depth treatment of the subject, the reader is referred to several excellent recent reviews [27,46,47].