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 ЄADPR in neutral aqueous solution is dynamically quenched by both quenching agents. The quenching of free ЄNAD+ by methionine was found to be predominantly static and was satisfactorily described to result from complex formation between quencher and dinucleotide. The rate constant for quenching by iodide of ЄNAD+ in the ternary complex with LADH and pyrazole is comparable to that of free ЄADPR or ЄADP. It is concluded that the bound Є-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 (ADPR to GPDH, ЄNAD+ to LDH, and oxalate to the LDH:ЄNAD+ 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. © 1979, American Chemical Society. All rights reserved.