Analysis of purified maize starch synthases IIa and IIb: SS isoforms can be distinguished based on their kinetic properties

Since starch synthases IIa (SSIIa) and SSIIb have not been purified from pl ant tissue, their structure-function relationships have not keen well chara cterized. Therefore, we have expressed these SS genes in Escherichia coli, purified them to apparent homogeneity, and studied their kinetic properties . In addition, the N-terminally truncated forms of these enzymes mere studi ed in an attempt to understand the function of the diverse N-terminal seque nces in SS. Our results show that, Like SSI, the N-terminal extensions of S SIIa and SSIIb are not essential for catalytic activity and no extensive ch anges in their kinetic properties are observed upon their N-terminal trunca tion. Each isoform of SS can be distinguished based on its kinetic properti es. Maize SSI and maize SSIIb exhibit higher V-max with glycogen as a prime r, while the converse is true for SSIIa. However, the specific activity of SSIIb is at least two- to threefold higher than that for either SSI or SSII a. Although-SSIIb exhibits the highest maximal velocity of the isoforms com pared, its apparent affinity for primer is twofold lower than the affinity of SSI and SSIIa for primer. Perhaps these differences in primer affinity, primer preference, and maximal velocities all contribute in some way to the different structure(s) of starch during its synthesis. Expression and puri fication of maize SS has now provided us a useful tool to address the role( s) of SS in starch synthesis and starch structure. (C) 1999 Academic Press.