Tautomerism of 2-azidoadenine nucleotides Effects on enzyme kinetics and photoaffinity labeling

• Published in: Biochimica et biophysica acta Vol. 800; no. 1; pp. 41 - 51
• Main Author: Czarnecki, Joseph J.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 16.07.1984; Elsevier; North-Holland
• Subjects: Adenosine Diphosphate - analogs & derivatives; Adenosine Diphosphate - chemical synthesis; Adenosine Diphosphate - metabolism; Adenosine Monophosphate - analogs & derivatives; Adenosine Monophosphate - chemical synthesis; Adenosine Monophosphate - metabolism; Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - chemical synthesis; Adenosine Triphosphate - metabolism; Adenylate Kinase - metabolism; Affinity Labels - chemical synthesis; Analytical, structural and metabolic biochemistry; Animals; Azides - chemical synthesis; Azides - metabolism; Azidoadenine nucleotide; Biological and medical sciences; Cattle; Enzyme kinetics; Fundamental and applied biological sciences. Psychology; Hexokinase - metabolism; Kinetics; Molecular Conformation; Muscles - enzymology; Myocardium - enzymology; Nucleic acids; Nucleic bases, nucleotides; Phosphofructokinase-1 - metabolism; Phosphotransferases - metabolism; Photoaffinity labeling; Proton-Translocating ATPases - metabolism; Pyruvate Kinase - metabolism; Rabbits; Saccharomyces cerevisiae - enzymology; Substrate Specificity; Swine; Tautomerism; Photoaffinity labeling; Azidoadenine nucleotide; 2-N 3 ADP; 8-BrAMP; 8-BrADP; 2-N 3AMP; 8-azido AMP; Tautomerism; Enzyme kinetics; 2-N 3ATP; 1-Phosphofructokinase; Adenylate kinase; Pyruvate kinase; Enzyme; Affinity labelling; Analog; Molecular interaction; Binding site; Kinetics; Adenine nucleotide; Conformation
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/0304-4165(84)90092-8

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 F 1-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°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.