Human pancreatic glucokinase. Structural and physico-chemical studies related to catalytic activation, kinetic cooperativity and GCK-diabetes.

Abstract

Glucokinase (GK) functions as a glucose sensor in insulin-producing pancreatic-cells and as a regulator of hepatic glycolysis, glycogen synthesis and gluconeogenesis. Its key role in glucose homeostasis is evidenced by naturally occurring GK gene mutations causing monogenic diabetes and hyperinsulinemic hypoglycemia and by the discovery of allosteric GK activators (GKA) that hold promise as new antidiabetic agents. GK catalyzes the first step in glucose metabolism, i.e. the conversion of -D-glucose to glucose-6-phosphate (G6P), using MgATP2- as the phosphoryl donor. Glucose activates GK on its binding to the active site by inducing a global conformational change. Using intrinsic tryptophan fluorescence (ITF) spectroscopy as a probe on the glucose-induced conformational change, we identified key residues in this process. The glucose-induced fluorescence increase was primarily determined by W99 and W167, and little affected by W257. Based on results from functional mutagenesis and structural dynamic analyses, we have proposed that three active site residues (N204, N231 and E256) in the L-domain function as primary contact residues for glucose binding to the super-open form. Moreover, local torsional stresses at N204 and D205 of the highly flexible connecting region II was important for the subsequent propagation of the conformational transition towards cleft closure. No structural data have been available on ATP binding to the apoenzyme and how it possibly affects its conformation. Here, we provide the first experimental evidence for an equilibrium binding of ATP and its analogue AMP-PNP to the ligand-free enzyme. Moreover, ITF quenching analyses and molecular dynamics (MD) simulations indicated a significant conformational change upon nucleotide binding. This finding was supported by the protective effect of ATP on binding of the extrinsic fluorescence probe ANS and on limited proteolysis with trypsin. Furthermore, the modeled structure of the GK-ATP binary complex provided insight into the active site contact residues involved in the interaction with ATP. The knowledge on covalent modifications of human GK (hGK) and their possible regulatory functions are limited, and the molecular and cellular mechanisms involved in its degradation/turnover are also poorly understood. Using the rabbit reticulocyte lysate (RRL) as an in vitro model system, we demonstrated that pancreatic-cell (isoform 1) and liver (isoform 2) hGK are substrates for the ubiquitin-conjugating enzyme system, and that both isoforms are polyubiquitinated on at least two lysine residues. A putative ubiquitin interacting motif (UIM) site at the C-terminal end was identified by 3D structural analysis, and associated with polyubiquitination at one of the sites. Moreover, our results supported that poly/multiubiquitination of recombinant pancreatic hGK in vitro target the newly synthesized enzyme for proteasomal degradation. Interestingly, purified free pentaubiquitin chains were demonstrated to interact with and allosterically activate (~1.4-fold) recombinant hGK, assigned to their equilibrium binding to the UIM site. Both these ubiquitin-mediated processes represent potential physiological regulatory mechanisms of GK.