Accessibility of adenine binding sites in dehydrogenases to small molecules studied by fluorescence quenching.
Quenching of the fluorescence of ethenoadenine derivatives by iodide ions and by methionine was studied in solution and when the nucleotides were bound to several dehydrogenases. The fluorescence of epsilonADPR in neutral aqueous solution is dynamically quenched by both quenching agents. The quenching of free epsilonNAD+ by methionine was found to be predominantly static and was satisfactorily described to result from complex formation between quencher and dinucleotide. The rat constant for quenching by iodide of epsilonNAD+ in the ternary complex with LADH and pyrazole is comparable to that of free epsilonADPR or epsilonADP. it is concluded that the bound epsilon-adenine ring is partially exposed to the solvent. The opening, to the solvent, of the adenine binding site is not large enough to allow free methionine diffusion since the rate constant for quenching of bound coenzyme by this quenching agent is relatively small. The difference between the rate constants for quenching of free and enzyme bound nucleotide was used to evaluate the binding constants of epsilonADPR to GPDH, epsilonNAD+ to LDH, and oxalate to the LDH:epsilonNAD+ complex. This technique may prove to be particularly useful when the binding of a fluorescent ligand to a protein is not accompanied by significant changes in its fluorescence.
D000227:Adenine Nucleotides; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives; D000247:Adenosine Diphosphate Sugars; D000429:Alcohol Oxidoreductases; D001665:Binding Sites; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D007700:Kinetics; D007770:L-Lactate Dehydrogenase; D008433:Mathematics; D008715:Methionine; D009243:NAD / Q000031:analogs & derivatives; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D000227:Adenine Nucleotides; D000247:Adenosine Diphosphate Sugars; D009243:NAD; D000244:Adenosine Diphosphate; D008715:Methionine; D000429:Alcohol Oxidoreductases; D007770:L-Lactate Dehydrogenase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases