Adenosine kinase from human erythrocytes: kinetic studies and characterization of adenosine binding sites.
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.
D000241:Adenosine / Q000378:metabolism; D000248:Adenosine Kinase / Q000097:blood* / Q000302:isolation & purification; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001665:Binding Sites; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D010770:Phosphotransferases / Q000097:blood*; D011485:Protein Binding
D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D004228:Dithionitrobenzoic Acid; D010770:Phosphotransferases; D000248:Adenosine Kinase; D000241:Adenosine