Structural basis for ADP-dependent glucokinase inhibition by 8-bromo–substituted adenosine nucleotide

Abstract

In higher eukaryotes, several ATP-utilizing enzymes known as hexokinases activate glucose in the glycolysis pathway by phosphorylation to glucose 6-phosphate. In contrast to canonical hexokinases, which use ATP, ADP-dependent glucokinase (ADPGK) catalyzes noncanonical phosphorylation of glucose to glucose 6-phosphate using ADP as a phosphate donor. Initially discovered in Archaea, the human homolog of ADPGK was described only recently. ADPGK's involvement in modified bioenergetics of activated T cells has been postulated, and elevated ADPGK expression has been reported in various cancer tissues. However, the physiological role of ADPGK is still poorly understood, and effective ADPGK inhibitors still await discovery. Here, we show that 8-bromo-substituted adenosine nucleotide inhibits human ADPGK. By solving the crystal structure of archaeal ADPGK in complex with 8-bromoadenosine phosphate (8-Br-AMP) at 1.81 Å resolution, we identified the mechanism of inhibition. We observed that 8-Br-AMP is a competitive inhibitor of ADPGK and that the bromine substitution induces marked structural changes within the protein's active site by engaging crucial catalytic residues. The results obtained using the Jurkat model of activated human T cells suggest its moderate activity in a cellular setting. We propose that our structural insights provide a critical basis for rational development of novel ADPGK inhibitors.

Crystal Structure of Kluyveromyces lactis Glucokinase (KlGlk1)

“…In Kl Hxk1, the substrate binding promotes the closed conformation of protein, whereas apo Kl Glk1 remains in open state. This phenomenon is typical for sugar phosphorylating proteins and has been described before [5,31]. The difference in protein conformation and domain location supports our suggestion concerning Kl Glk1 open state.…”

“…Kl Glk1 activity was assessed by monitoring the level of one of the reaction products (glucose-6-phosphate) in a coupled glucose-6-phosphate dehydrogenase (G6PD) reaction essentially as described previously [5,6]. Unless stated otherwise, the assays were performed at 37 °C in reaction buffer (20 mM Tris-HCl pH 7.4, 250 mM sucrose, 50 mM KCl, 5 mM MgCl 2 ) supplemented with glucose-6-phosphate dehydrogenase (1 unit) and nicotinamide adenine dinucleotide phosphate (NADP) (0.5 mM).…”

“…Glucose phosphorylation is also considered as drug target against parasitic protists [1,2,3]. Additionally, some ADP-dependent sugar kinases were also characterized, but their role remains more elusive [4,5,6].…”

Structural Characterization of Glycerol Kinase from the Thermophilic Fungus Chaetomium thermophilum

“…To uncover the structural determinants of nucleotide-binding by CtGK, we attempted to crystalize either binary CtGK–ATP or ternary CtGK–ATP–glycerol complexes. Despite significant efforts, including co-crystallization or ligand soaking procedures, and testing the known nucleotide-mimicking compounds such as 8-Br-AMP, 5-ITU, or AMPNP [ 18 , 28 , 29 ], no electron density allowing for reliable nucleotide modeling was identified in any of the analyzed crystals. However, taking into account the high structural similarity between CtGK and PfGK, especially in the active site, we can infer the most likely position of the ATP by comparison of these two structures.…”

“…MjADPGK and phADPGK/PFK were obtained as previously described [ 18 , 28 ]. Briefly, genes encoding full-length mjADPGK and phADPGK were synthesized (GenScript, Leiden, Netherlands) and cloned into pET24d and pET15b plasmid, respectively.…”