Studies on the changes in protein fluorescence and enzymic activity of aspartate aminotransferase on binding of pyridoxal 5'-Phosphate

Abstract

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 of the number ofcofactors bound to the dimer. 5. The /Jsubformhad approximately 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 glutamate, 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 subform) decreased linearly, showing that each dimeric enzyme molecule contained one active and one inactive bound cofactor. 7. The results are not consistent with a flip-flop mechanism for this enzyme.