title: Tautomerism of 2-azidoadenine nucleotides. Effects on enzyme kinetics and photoaffinity labeling.
abstract: The 2-azidoadenine nucleotides show promise as photoaffinity probes. Substitution at the C-2 position should favor an anti conformation and enable binding of the analogue to enzyme sites which exhibit low affinity for the 8-azidoadenine derivatives. The 2-azidoadenine nucleotides were found to be substrates for pyruvate kinase, phosphofructokinase, adenylate kinase, hexokinase and the mitochondrial F1-ATPase. However, tautomerism of 2-azidoadenine nucleotides to two nonphotoreactive tetrazole forms complicates kinetic analyses and their use as photoaffinity probes. An analysis of the ultraviolet spectra of these analogues enables an estimation of the tetrazolo isomer content and the rates of tautomerization. The photoreactive azido isomer was found to represent only 45% of the total analogue population in neutral aqueous solution. The azidoazomethine-tetrazole equilibrium favors the azido isomer in acidic or nonpolar solutions. The first-order rate constants at 25 degrees C were determined to be 0.017 min-1 and 0.021 min-1 for tautomerism to the azido and tetrazolo isomers, respectively. Prior equilibration of the probe in various solvents thus allows investigation of the analogue's behavior with an enzyme system at different, essentially fixed, isomer ratios. The determination of the impact of the tetrazolo tautomers on the system allows optimization of conditions for photoaffinity-labeling experiments.
mesh_terms: D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D000263:Adenylate Kinase / Q000378:metabolism*; D000345:Affinity Labels / Q000138:chemical synthesis*; D000818:Animals; D001386:Azides / Q000138:chemical synthesis* / Q000378:metabolism; D002417:Cattle; D006593:Hexokinase / Q000378:metabolism*; D007700:Kinetics; D008968:Molecular Conformation; D009132:Muscles / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011770:Pyruvate Kinase / Q000378:metabolism*; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity; D013552:Swine
chemical_list: D000345:Affinity Labels; D001386:Azides; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; C022295:2-azidoadenosine 3',5'-diphosphate; C018838:2-azidoadenosine 5'-monophosphate; C042231:2-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D006593:Hexokinase; D010732:Phosphofructokinase-1; D011770:Pyruvate Kinase; D000263:Adenylate Kinase; D006180:Proton-Translocating ATPases