N- and C-terminal peptide sequences are essential for enzyme assembly, allosteric, and/or catalytic properties of ADP-glucose pyrophosphorylase.

Abstract

ADP-glucose pyrophosphorylase is a key regulatory enzyme in starch synthesis in most plant tissues. Unlike the allosteric regulatory dependent properties of the leaf enzyme, the enzymes from non-photosynthetic tissues exhibit varying levels of sensitivity to allosteric regulation, a behavior which may be an inherent property of the enzyme or a product of post-translational modification. As partial proteolysis of the holoenzyme may account for the wide variation of allosteric regulatory behavior exhibited by enzymes from non-photosynthetic tissues, small N- and C-terminal peptide deletions were made on either the potato large and small subunit and co-expressed with the counterpart wild-type subunit in Escherichia coli. Removal of the putative carboxy-terminal allosteric binding region from either subunit type results in an abolishment of enzyme formation indicating that the carboxy terminus of each subunit type is essential for proper subunit folding and/or enzyme assembly as well as its suggested role in allosteric regulation. Removal of a small 10 amino acid peptide from the N-terminus of the small subunit increased its resistance to the allosteric inhibitor Pi as well as its sensitivity to heat treatment. Likewise, removal of the corresponding peptide (17 residues) at the N-terminus of the large subunit also increased its resistance towards Pi inhibition but, in addition, increased its sensitivity to 3-PGA activation. Deletion of an additional 11 residues reversed these changes in allosteric properties but at the expense of a reduced catalytic turnover rate. Combined, these results indicate that the N- and C-terminal regions are essential for the proper catalytic and allosteric regulatory properties of the potato ADP-glucose pyrophosphorylase. The possible significance of these results on the observed insensitivity to effector molecules by ADP-glucose pyrophosphorylases from other non-photosynthetic tissues is discussed.