Structure of Human Adenosine Kinase at 1.5 Å Resolution^,

Abstract

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

Crystal Structure of Brain Pyridoxal Kinase, a Novel Member of the Ribokinase Superfamily

“…All of the other kinases contain a lid structure covering the bound substrates. In ribokinase (22), adenosine kinase (24,34) and ADP-dependent glucokinase (36), a small domain of the enzyme structure forms this lid structure. However, in the trimeric structure of THZ kinase, each monomer plays the role of the lid for the adjacent monomer (35), and in HMPP kinase, a nascent lid formed by a flexible loop and two ␤-strands exists (25).…”

Synthesis, biological evaluation and molecular modeling studies of N6-benzyladenosine analogues as potential anti-toxoplasma agents

“…Specifically, the activity of adenosine kinase (EC.2.7.1.20), the enzyme responsible for the salvage of adenosine in this parasite, is 10-fold higher than those of other enzymes in the purine salvage pathways, and hence contributes more significantly to the salvage of purines in T. gondii than any other enzyme of the purine salvage pathways [12][13][14][15][16]. Secondly, structure-activity relationships [17,18], comparative enzymatic [18][19][20], metabolic [19][20][21], molecular [22], and X-ray structural studies [23][24][25] have demonstrated that there are sufficient differences between the active sites and substrate specificities of human and T. gondii adenosine kinases that can be exploited to design specific "subversive substrates" for the T. gondii enzyme [12,13].…”

Expression in Escherichia coli of a recombinant adenosine kinase from Saccharomyces cerevisiae: purification, kinetics and substrate analyses

“…In addition, the crystal structures of bacterial ribokinase (RK) (Andersson and Mowbray, 2002), Toxoplasma gondii AK (Schumacher et al, 1999) and human AK (Mathews et al, 1998) have been solved and found to be notably similar. These structure analyses have provided valuable insights into substrate binding sites and reaction mechanisms.…”