The Structural and Functional Characterization of Mammalian ADP-dependent Glucokinase.

The enzyme-catalyzed phosphorylation of glucose to glucose-6-phosphate is a reaction central to the metabolism of all life. ADP-dependent glucokinase (ADPGK) catalyzes glucose-6-phosphate production, utilizing ADP as a phosphoryl donor in contrast to the more well characterized ATP-requiring hexokinases. ADPGK is found in Archaea and metazoa; in Archaea, ADPGK participates in a glycolytic role, but a function in most eukaryotic cell types remains unknown. We have determined structures of the eukaryotic ADPGK revealing a ribokinase-like tertiary fold similar to archaeal orthologues but with significant differences in some secondary structural elements. Both the unliganded and the AMP-bound ADPGK structures are in the "open" conformation. The structures reveal the presence of a disulfide bond between conserved cysteines that is positioned at the nucleotide-binding loop of eukaryotic ADPGK. The AMP-bound ADPGK structure defines the nucleotide-binding site with one of the disulfide bond cysteines coordinating the AMP with its main chain atoms, a nucleotide-binding motif that appears unique to eukaryotic ADPGKs. Key amino acids at the active site are structurally conserved between mammalian and archaeal ADPGK, and site-directed mutagenesis has confirmed residues essential for enzymatic activity. ADPGK is substrate inhibited by high glucose concentration and shows high specificity for glucose, with no activity for other sugars, as determined by NMR spectroscopy, including 2-deoxyglucose, the glucose analogue used for tumor detection by positron emission tomography.

Expression and role in glycolysis of human ADP-dependent glucokinase.

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.