| Abstract
| - ADPglucose pyrophosphorylase catalyzes the regulatory step in the pathway for bacterialglycogen synthesis. The enzymes from different organisms exhibit distinctive regulatory properties relatedto the main carbon metabolic pathway. Escherichia coli ADPglucose pyrophosphorylase is mainly activatedby fructose 1,6-bisphosphate (FBP), whereas the Agrobacterium tumefaciens enzyme is activated by fructose6-phosphate (F6P) and pyruvate. Little is known about the regions determining the specificity for theallosteric regulator. To study the function of different domains, two chimeric enzymes were constructed.“AE” contains the N-terminus (271 amino acids) of the A. tumefaciens ADPglucose pyrophosphorylaseand the C-terminus (153 residues) of the E. coli enzyme, and “EA”, the inverse construction. Expressionof the recombinant wild-type and chimeric enzymes was performed using derivatives of the pET24a plasmid.Characterization of the purified chimeric enzymes showed that the C-terminus of the E. coli enzyme isrelevant for the selectivity by FBP. However, this region seems to be less important for the specificity byF6P in the A. tumefaciens enzyme. The chimeric enzyme AE is activated by both FBP and F6P, neitherof which affect EA. Pyruvate activates EA with higher apparent affinity than AE, suggesting that theC-terminus of the A. tumefaciens enzyme plays a role in the binding of this effector. The allosteric inhibitorsite is apparently disrupted, as a marked desensitization toward AMP was observed in the chimeric enzymes.
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