Patates ADP glikoz pirofosforilaz enziminin allosterik özelliklerinin modulasyonu için önemli amino asitlerin belirlenmesi

ADP glucose pyrophosphorylase (AGPase) is a key regulatory enzyme of bacterial glycogen and plant starch synthesis as it controls carbon flux via its allosteric regulatory behavior. Whereas the bacterial enzyme is composed of a single subunit type, the plant AGPase is a heterotetrameric enzyme (?2ß2) with distinct roles for each of the two subunit types. The large subunit (LS) is involved mainly in allosteric regulation through its interaction with the catalytic small subunit (SS). Previously, critical amino acids of potato (Solanum tuberosum L.) LS that interact with SS in the native heterotetramer structure were identified both computationally and experimentally. In this study, we aimed to improve the heterotetrameric assembly of potato AGPase and to detect residues located on the interface involving the allosteric regulation of the enzyme with a reverse genetics approach. A mutant, ?2ß2 formation deficient, large subunit of potato AGPase named LSR88A was subjected to random mutagenesis using error prone PCR and screened for the capacity to form an enzyme restoring glycogen production in glgC- Escherichia coli, AGPase activity deficient, containing wild type SS by assessing iodine staining. Fifteen suppressor mutants were identified and sequence analysis of these mutants revealed that mutations are mainly clustered at subunit interface and nearby the subunit interface. Subsequently, R88A mutation was reversed with site directed mutagenesis to see the effect of these mutations in the absence of R88A mutation. Kinetic characterization showed that two random mutants, named RM2 and RM10, exhibit altered allosteric properties than the wild type. These results indicate that interfaces between the large and small subunits are significant for the allosteric properties of the AGPase. Obtaining stable and up-regulated AGPase variants will enable us to use these mutants to increase the starch yield in crop plants.