Adenosine kinase from human erythrocytes: kinetic studies and characterization of adenosine binding sites

Abstract

The reaction catalyzed by adenosine kinase purified from human erythrocytes proceeds via a classical ordered sequential mechanism in which adenosine is the first substrate to bind to and AMP is the last product to dissociate from the enzyme. However, the interpretation of the steady-state kinetic data is complicated by the finding that while AMP acts as a classical product inhibitor at concentrations greater than 5 mM, at lower concentrations AMP can act as an apparent activator of the enzyme under certain conditions. This apparent activation by AMP is proposed to be due to AMP allowing the enzyme mechanism to proceed via an alternative reaction pathway that avoids substrate inhibition by adenosine. Quantitative studies of the protection of the enzyme afforded by adenosine against both spontaneous and 5,5'-dithiobis(2-nitrobenzoic acid)-mediated oxidation of thiol groups yielded "protection" constants (equivalent to enzyme-adenosine dissociation constant) of 12.8 microM and 12.6 microM, respectively, values that are more than an order of magnitude greater than the dissociation constant (Kia = 0.53 microM) for the "catalytic" enzyme-adenosine complex. These results suggest that adenosine kinase has at least two adenosine binding sites, one at the catalytic center and another quite distinct site at which binding of adenosine protects the reactive thiol group(s). This "protection" site appears to be separate from the nucleoside triphosphate binding site, and it also appears to be the site that is responsible for the substrate inhibition caused by adenosine.

Structure of Human Adenosine Kinase at 1.5 Å Resolution,

“…Since the adenosine 1 binding site is significantly more buried and further from the solvent interface than adenosine 2, it seems reasonable to postulate that, without invoking a protein conformational change, adenosine binds prior to ATP. This is consistent with most of the earlier kinetic studies (21)(22)(23)(24) but not all (20).…”

“…Our studies confirm the presence of both binding sites and suggest that the substrate inhibition is due to competitive inhibition of ATP binding. It should be noted, however, that the latter conclusion is inconsistent with other kinetic data showing adenosine to be a noncompetitive inhibitor of ATP binding (21) 1 and with conclusions drawn from DTNB inactivation studies suggesting that the ATP-binding site is distinct from the two adenosine sites (23). These conclusions were based primarily on the failure of ATP to inhibit AK inactivation by DTNB, whereas inactivation was prevented by adenosine but only at concentrations an order of magnitude greater than the estimated dissociation constant for adenosine at the catalytic site.…”

“…Studies with partially purified adenosine kinase from Ehrlich ascites tumor cells identified ATP as the first substrate to bind and AMP as the last product to be released (20). Other studies, however, predicted adenosine to be the first substrate to bind (21)(22)(23)(24) while still others predicted ADP to be the last substrate to leave (15).…”

“…Saturation kinetics suggested two adenosine-binding sites; one with high affinity which corresponded to the catalytic site and one with much lower affinity. The latter site was reported to differ from the triphosphate-binding site based on competition studies and may serve to regulate AK activity during times of ischemia and high adenosine production (23,25,26). Chemical modification studies supported the existence of a second adenosine-binding site distinct from the ATP site.…”

Mutational analysis of the active-site residues crucial for catalytic activity of adenosine kinase from Leishmania donovani

“…Besides phosphorylating Ado, the enzyme also recognizes a wide variety of Ado analogues as substrates, thus making it a prospective target for chemotherapy [1]. Although the enzyme was kinetically characterized from a number of sources, including higher eukaryotes [2][3][4], parasitic protozoa [5][6][7][8], plants [9] and bacteria [10], the mechanism of substrate recognition and phosphorylation of various nucleosides/analogues by this enzyme was the subject of substantial debate until recently. Using 6-methylmercaptopurine riboside as the substrate, Henderson et al [11] claimed that the enzyme from Ehrlich ascites tumour cells carried out a reaction in which ATP was the first substrate to bind and 6-methylmercaptopurine riboside monophosphate was the last product to be released.…”