Glutamik asit 358, patates adp-glikoz pirofosforilazının normal allosterik fonksiyonu ve heterotetramer oluşumu için önemlidir

ADP-glucose pyrophosphorylase (AGPase) is a key enzyme in plant starch biosynthesis. It contains large (LS) and small (SS) subunits encoded by two different genes. Experimental and computational studies indicated that interfacial amino acids in potato AGPase are important for the subunit-subunit interactions and allosterism. Among identified amino acid residues; A91, F101, F311 and E358 on LS (identified through error prone PCR) was found to be potent for influencing the allosteric and the catalytic properties potato AGPase. In this study, effect of these mutations on heterotetramer assembly and allosteric and catalytic properties of potato AGPase were investigated. All mutations were applied on to wild type (WT) potato AGPase large subunit cDNA using site directed mutagenesis PCR. Mutants LS, together with WT-LS, was transformed to E.coli glgC- containing the potato SS cDNA plasmid, that are deficient in glycogen production. All the LS mutants except LS-E358G were able complement glgC- in E.coli. LS-E358G mutant subjected to the detailed biochemical characterization to investigate its catalytic allosteric and structural properties. Compared to WT AGPase, AGPase with mutant LS-E358G was found to have a different assembly profile, having more heterotetramers and less monomers and dimers. Its regulatory properties were noticeably altered, its affinity towards its activator reduced and inhibitor increased. Interestingly, although large subunit of potato AGPase is mostly regulatory, this mutation considerably reduced the affinity of the AGPase towards its reverse and forward direction substrates ATP and ADP-glucose, showing potato tuber AGPase large subunit affects the catalytic performance of enzyme.