Abstract
The allosteric enzyme ADPGlucose Pyrophosphorylase (ADPG PPase) catalyzes the rate‐limiting step in glucan biosynthesis. The enzyme from Rhodobacter sphaeroides is perhaps the most highly regulated ADPG PPase and is activated by F6P, FBP, and pyruvate. Given the diverse sequences present in the ADPG PPase family at the N‐terminus and the lack of structural information about this region, it was of interest to probe the role of the extreme N‐terminus by truncation mutagenesis. Three truncation constructs have been generated (initiating at the third, fifth, and ninth codons) and the altered proteins have been successfully expressed and purified to homogeneity using anion exchange, hydrophobic interaction, and Cibacron Blue chromatography. Truncation 1 displayed a Vmax ~20% of wild‐type and S0.5 and A0.5 values for substrates and activators ~2–3 fold higher than wild type with ~2‐fold higher activation by activators. Truncation 3 exhibited only ~1% of wild type activity and also an extremely diminished response to activators. The N‐terminus is clearly important for optimal activity and regulation. Complete characterization of the altered proteins is in progress. Supported in part by NSF grant 0448676.
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