Investigation of subunit function in ADP-glucose pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase), a hey regulatory enzyme in higher p lant starch biosynthesis, is composed of a pair of large and small subunits (alpha (2)beta (2)). Current evidence suggests that the large subunit has primarily a regulatory function, while the small subunit has both regulator y and catalytic roles. To define the structure-function relationship of the large subunit (LS), the LS of potato AGPase was subjected to chemical muta genesis and coexpressed with the wild-type (WT) small subunit (SS) cDNA in an AGPase defective Escherichia coli strain. An LS mutant (M143) was isolat ed, which accumulated very low levels of glycogen compared to the WT recomb inant AGPase, but maintained normal catalytic activity when assayed under s aturating conditions. Sequence analysis revealed that M143 has a single ami no acid change, V463I, which lies adjacent to the C-terminus. This single m utation had no effect on the Km for ATP and Mg2+, which were similar to the WT enzyme. The K-m for glucose 1-P, however, was sixfold higher than the W T enzyme. These results suggest that the LS plays a role in binding glucose 1-P through its interaction with the SS. (C) 2001 Academic Press.