Essential arginine residues in maize starch synthase IIa are involved in both ADP-glucose and primer binding.

The arginine-specific reagent phenylglyoxal inactivated the activity of maize starch synthase IIa (SSIIa), due to the modification of at least one arginine residue out of a possible 42. The addition of ADPGlc completely protected SSIIa from the inactivation, indicating that arginine may be involved in the interaction of this anionic substrate with SSIIa. However, site-directed mutagenesis of the conserved Arg-214 in SSIIa showed that this amino acid is important for apparent affinity of SSIIa for its primer (amylopectin and glycogen), as evidenced by a marked increase in the K(m) for primer upon substitution of this amino acid with no concomitant change in V(max), K(m) for ADPGlc, or secondary structure. Therefore, Arg-214 of SSIIa appears to play a role in its primer binding.

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 plant tissue, their structure-function relationships have not been well characterized. 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 were studied in an attempt to understand the function of the diverse N-terminal sequences in SS. Our results show that, like SSI, the N-terminal extensions of SSIIa and SSIIb are not essential for catalytic activity and no extensive changes in their kinetic properties are observed upon their N-terminal truncation. Each isoform of SS can be distinguished based on its kinetic properties. Maize SSI and maize SSIIb exhibit higher Vmax with glycogen as a primer, 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 SSIIa. Although SSIIb exhibits the highest maximal velocity of the isoforms compared, 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 purification of maize SS has now provided us a useful tool to address the role(s) of SS in starch synthesis and starch structure.

Purification and characterization of maize starch synthase I and its truncated forms.

Comparison of the protein sequences deduced from the cDNAs of maize granule-bound starch synthase, Escherichia coli glycogen synthase, and maize starch synthase I (SSI) reveals that maize SSI contains an N-terminal extension of 93 amino acids. In order to study the properties of maize SSI and to understand the functions of the maize SSI N-terminal extension, the gene coding for full-length SSI (SSI-1) and genes coding for N-terminally truncated SSI (SSI-2 and SSI-3) were individually expressed in E. coli. Here we describe for the first time the purification of a higher plant starch synthase to apparent homogeneity. Its kinetic properties were therefore studied in the absence of interfering amylolytic enzymes. The specific activities of the purified SSI-1, SSI-2, and SSI-3 were 22.5, 33.4, and 26.3 micromol Glc/min/mg of protein, respectively, which are eight times higher than those of partially purified SSI from developing maize endosperm. The full-length recombinant enzyme SSI-1 exhibited properties similar to those of the enzyme from maize endosperm. As observed for native maize enzyme, recombinant SSI-1 exhibited "unprimed" activity without added primer in the presence of 0.5 M citrate. Our results have clearly indicated that the catalytic center of SSI is not located in its N-terminal extension. However, N-terminal truncation decreased the enzyme affinity for amylopectin, with the Km for amylopectin of the truncated SSI-3 being about 60-90% higher than that of the full-length SSI-1. These results suggest that the N-terminal extension in SSI may not be directly involved in enzyme catalysis, but may instead regulate the enzyme binding of alpha-glucans. Additionally, the N-terminal extension may play a role in determining the localization of SSI to specific portions of the starch granule or it may regulate its interactions with other enzymes involved in starch synthesis.