抄録
The process of reconstitution of potato apophosphorylase with pyridoxal 5'-phosphate (PLP) has been investigated to elucidate the structure-function relationship in phosphorylase [EC 2. 4. 1. 1]. In time-course studies, the recovery of enzyme activity was found to be delayed, especially at low temperatures, compared with the binding of PLP to protein. On polyacrylamide gel electrophoresis, an intermediary enzyme species was detected which bound PLP in the same binding mode as the native holoenzyme does, but which had neither enzyme activity nor affinity for the substrate amylopectin. The intermediate is monomeric and is converted to the active holoenzyme with concomitant dimerization. NaBH4-treatment of the monomeric intermediate produced the reduced monomeric enzyme, which could be converted into the reduced dimeric enzyme with considerable enzyme activity. The findings support the view that the catalytic activity of phosphorylase requires the dimeric structure of protein. As in the animal enzyme, the aldimine bond between the PLP and the ε-amino group of the lysyl residue is also not essential for enzyme activity in plant phosphorylase.
