Expression and role in glycolysis of human ADP-dependent glucokinase

Abstract

A novel murine enzyme, ADP-dependent glucokinase (ADPGK), has been shown to catalyse glucose phosphorylation using ADP as phosphoryl donor. The ancestral ADPGK gene appears to have been laterally transferred from Archaea early in metazoan evolution, but its biological role has not been established. Here, we undertake an initial investigation of the functional properties of human ADPGK in human tumour cell lines and specifically test the hypothesis that ADPGK might prime glycolysis using ADP under stress conditions such as hypoxia. Recombinant human ADPGK was confirmed to catalyse ADP-dependent glucose phosphorylation in vitro, with an apparent K (M) for glucose of 0.29 mM. Expression databases and western blotting of surgical samples demonstrated high expression in many human tissues, including tumours. Unlike hexokinase-2 (HK2), RNAi studies with exon arrays showed that ADPGK is not a transcriptional target of hypoxia inducible factor-1. Consistent with this, ADPGK protein was not upregulated by hypoxia or anoxia. Surprisingly, stable fivefold overexpression of ADPGK in H460 or HCT116 cells had no apparent effect on proliferation or glycolysis, and did not rescue clonogenicity or glycolysis when HK2 was suppressed by siRNA. Furthermore, suppression of ADPGK by siRNA did not cause detectable inhibition of glycolysis or cell killing by anoxia, although it did induce a statistically significant decrease in plating efficiency of H460 cells under aerobic conditions. Thus, human ADPGK catalyses ADP-dependent phosphorylation of glucose in vitro, but despite its high expression in human tumour cell lines it appears not to make a quantifiable contribution to glycolysis under the conditions evaluated.

Mitochondria as Oxidative Signaling Organelles in T-cell Activation: Physiological Role and Pathological Implications

“…ADPGK mediates the first step of glycolysis by phosphorylating glucose using ADP (Richter et al 2012;Ronimus and Morgan 2004). The enzyme with a previously unknown role for eukaryotic cells is typical for thermophylic Archaea (Guixe and Merino 2009).…”

The Structural and Functional Characterization of Mammalian ADP-dependent Glucokinase

“…ADPGK Kinetic Analysis-Human ADPGK enzymatic activity was measured in triplicate using a coupled assay with glucose-6-phosphate dehydrogenase as previously described (2,3). Assays contained 50 mM Bis-Tris-propane (pH 7.0 at 37°C), 100 mM KCl, 1 mM NADP ϩ , 1:1 molar ratio of ADP-MgCl 2 , 2 units Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (Sigma-Aldrich).…”

“…A relatively recently discovered ADP-dependent glucokinase (ADPGK) 3 (EC 2.7.1.147) catalyzes the phosphorylation of D-glucose to glucose-6-phosphate using MgADP as phosphoryl donor in contrast to the more typical ATP-utilizing hexokinases and glucokinases. ADPGK was first identified in Archaea, being involved in a modified Embden-Meyerhof glycolytic pathway (1), in which Archaea can also use an ADP-dependent phosphofructokinase (ADPPFK; EC 2.7.1.146).…”

“…ADPGK was first identified in Archaea, being involved in a modified Embden-Meyerhof glycolytic pathway (1), in which Archaea can also use an ADP-dependent phosphofructokinase (ADPPFK; EC 2.7.1.146). Bioinformatic analysis led to the identification of ADPGK in metazoa and the subsequent cloning and initial characterization of mammalian ADPGKs (2,3). Mammalian ADPGKs show modest sequence similarity to archaeal orthologues (ϳ20% amino acid identity).…”

“…Furthermore, ADPGK has been identified as a cholesterol binding protein in a proteomics screen (5). Despite the role of archaeal ADPGK in glycolysis, overexpression of ADPGK in H460 and HC116 human tumor cells showed no cell proliferative or glycolytic effects (3). ADPGK knock-out in these cell lines showed no detectable effect on glycolysis or extracellular acidification as assay end points, although O 2 consumption was generally lower for the ADPGK knock-out cells (6).…”