Printed in Great Britain Studies on the Changes in Protein Fluorescence and Enzymic Activity of Aspartate Aminotransferase on Binding of Pyridoxal 5'-Phosphate

1. The a and,B subforms of aspartate aminotransferase were purified from pig heart. 2. The a subform contained 2mol of pyridoxal 5'-phosphate. The apo-(a subform) could be fully reactived by combination with 2mol of cofactor. 3. The protein fluorescence of the apo-(a subform) decreased non-linearly with increase in enzyme activity and concentration of bound cofactor. 4. It is concluded that the enzyme activity/mol ofbound cofactor is largely independent ofthe number ofcofactors bound to the dimer. 5. The /Jsubformhad approxi-mately half the specific enzyme activity of the a subform, and contained an average of one active pyridoxal 5'-phosphate molecule per molecule, which could be removed by gluta-mate, and another inactive cofactor which could only be removed with NaOH. 6. On recombination with pyridoxal 5'-phosphate the protein fluorescence of the apo-(fl sub-form) decreased linearly, showing that each dimericenzyme moleculecontained one active and one inactive bound cofactor. 7. The results are not consistent with a flip-flop mecha-nism for this enzyme. It has been suggested that the tryptophan fluor-escence of oligomeric proteins will decrease non-linearly as successive ligand-binding sites on a given globular protein molecule are occupied by ligands that quench tryptophan fluorescence by long-distance radiationless transfer of electronic excitation energy (Holbrook, 1972). Non-linear quenching has been observed when fluorodinitrobenzene binds to avidin (Green, 1964), by di-iodotyrosines in serum albumin (Perlman et al., 1968), Fe3+ in transferrin (Lehrer, 1969), and NADH in oligomeric dehydrogenase