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.
Highlights
In higher eukaryotes, several ATP-utilizing enzymes known as hexokinases activate glucose in the glycolysis pathway by phosphorylation to glucose 6-phosphate
By solving the crystal structure of archaeal ADP-dependent glucokinase (ADPGK) in complex with 8-bromoadenosine phosphate (8-Br-AMP) at 1.81 Aresolution, we identified the mechanism of inhibition
ADPGK activity was monitored as the rate of glucose 6-phosphate formation measured in a coupled reaction with glucose-6-phosphate dehydrogenase, as described previously [3, 5, 10]
Summary
Biochemical characterization of hADPGK and identification of inhibitors hADPGK was expressed using an Escherichia coli recombinant system, and basic enzymatic properties were characterized. The specific activity of ADPGK with GDP as a substrate was only 6% of that determined under the same conditions in the presence of ADP as a phosphate donor (Fig. S1B). The initial activity of hADPGK in the presence of AMP was ϳ85% of the maximal enzymatic activity; this slight decrease was independent of AMP concentration up to 5 mM (Fig. 1C) allowing us to conclude that no significant product inhibition was associated with AMP at levels far exceeding the Km values for both substrates. To identify ADPGK inhibitors, several adenosine analogs were tested for their ability to reduce the recombinant hADPGK activity (Fig. 1C and Table 1, and Table S1). None of the tested analogs of 8-Br-AMP reduced the activity of hADPGK (even at 5 mM concentration) demonstrating the importance of the ␣-phosphate group in 8-bromo-substituted adenosine binding (Fig. 1C). Further evaluation demonstrated that 8-Br-AMP inhibition has a competitive nature (Fig. 1D and Fig. S2) and is characterized by Ki of 270 Ϯ 30 M
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