Abstract
Kinetics and structural studies of the plastidial Solanum tuberosum phosphorylase (stPho1) revealed that the most active form of the enzyme (stPho1ΔL78) is composed by two segments generated by proteolytic degradation of an approximately 65-residue-long peptide (L78) approximately in the middle of the stPho1 primary structure. stPho1ΔL78 is 1.5 times more active than the nonproteolyzed enzyme in solution and shows stronger specificity for glycogen, α-d-glucose, caffeine, and β-cyclodextrin than stPho1. The crystal structure of stPho1ΔL78 has been resolved at 2.2 Å resolution and revealed similarities and differences with the mammalian enzymes. The structural fold is conserved as is the active site, while other binding sites such as the inhibitor, the glycogen storage, the quercetin, and the allosteric are not. The binding of α-d-glucose, caffeine, and β-cyclodextrin to stPho1 has been studied by X-ray crystallography and revealed significant differences from those of the mammalian phosphorylases. As stPho1 is capable of catalyzing both starch synthesis and degradation, our studies suggest that the direction of stPho1 activity is regulated by the proteolytic degradation of the L78 peptide.