Adenosine kinase (AK) is a key enzyme in the regulation of extracellular adenosine and intracellular adenylate levels. Inhibitors of adenosine kinase elevate adenosine to levels that activate nearby adenosine receptors and produce a wide variety of therapeutically beneficial activities. Accordingly, AK is a promising target for new analgesic, neuroprotective, and cardioprotective agents. We determined the structure of human adenosine kinase by X-ray crystallography using MAD phasing techniques and refined the structure to 1.5 Å resolution. The enzyme structure consisted of one large R/ domain with nine -strands, eight R-helices, and one small R/ -domain with five -strands and two R-helices. The active site is formed along the edge of the -sheet in the large domain while the small domain acts as a lid to cover the upper face of the active site. The overall structure is similar to the recently reported structure of ribokinase from Escherichia coli [Sigrell et al. (1998) Structure 6, 183-193]. The structure of ribokinase was determined at 1.8 Å resolution and represents the first structure of a new family of carbohydrate kinases. Two molecules of adenosine were present in the AK crystal structure with one adenosine molecule located in a site that matches the ribose site in ribokinase and probably represents the substrate-binding site. The second adenosine site overlaps the ADP site in ribokinase and probably represents the ATP site. A Mg 2+ ion binding site is observed in a trough between the two adenosine sites. The structure of the active site is consistent with the observed substrate specificity. The active-site model suggests that Asp300 is an important catalytic residue involved in the deprotonation of the 5′-hydroxyl during the phosphate transfer.Adenosine kinase (ATP, adenosine 5′-phosphotransferase, EC 2.7.1.20) catalyzes the phosphorylation of ribofuranosylcontaining nucleoside analogues at the 5′-hydroxyl using ATP or GTP as the phosphate donor. Tissue distribution studies indicate that adenosine kinase (AK) is the most abundant nucleoside kinase in mammals with AK activity in humans and monkeys expressed at the highest levels in liver, kidney, and lung and at intermediate levels in the brain, heart, and skeletal muscle (1, 2). AK exhibits a relatively broad substrate specificity tolerating modifications in both the sugar and base moieties (3). Accordingly, numerous nucleoside antiviral and anticancer drugs are AK substrates and consequently undergo rapid phosphorylation in vivo to the 5′-monophosphate. In many cases, the monophosphate is subsequently converted by other kinases to the triphosphate which functions as the active metabolite. Examples include ribavirin (4) and mizoribine (5).The physiological function of AK is associated with the regulation of extracellular adenosine levels and the preservation of intracellular adenylate pools (6