Abstract
Pyridoxine deficiency caused a decrease in the amount of aromatic L-amino acid decarboxylase (AADC) in PC 12 cells to less than 5% of the control. The degree of the enzyme saturation with the coenzyme pyridoxal 5'-phosphate (PLP) was around 90% for both the control and the pyridoxine-deficient cells, contrary to earlier reports by others. Mathematical analysis of the result indicated that the AADC apoenzyme is degraded at least 20-fold faster than the holoenzyme in the cells. To determine the mechanism of the preferential degradation of the apoenzyme, in vitro model studies were carried out. AADC has a flexible loop that covers the active site. This loop was easily leaved by proteases at similar rates for both the holoenzyme and the apoenzyme. However, in the presence of the substrate analog, dopa methyl ester, the holoenzyme was not cleaved by proteases, while the apoenzyme was cleaved similarly. These results indicated that the ligand that forms a Schiff base (aldimine) with PLP is fixed to the active site and stabilizes the flexible loop. The structure of the rat AADC-dopa complex modeled on the crystal structure of pig AADC showed that the flexible loop can fit in the concave surface at the entrance of the active site, its aliphatic and aromatic residues forming hydrophobic interactions with the substrate catechol ring. It was postulated that the flexible loop of the holoenzyme is stabilized in vivo by taking a closed structure that holds the PLP-substrate aldimine, while the apoenzyme cannot bind the substrate and its flexible loop is easily cleaved, leading to the preferential degradation of the apoenzyme.
