REGULATORY MECHANISMS INVOLVED IN THE BIOSYNTHESIS OF STARCH

The reactions involved in starch synthesis have been shown to be catalyzed respectively, by ADPglucose synthetase, EC 2.7.7.27, (ATP + alpha-glucose 1-P <--> ADPglucose + PP(i)) and starch synthase, EC 2.4.1.21, (ADPGlc + alpha-1,4 glucan --> alpha-1,4 glucosly-alpha-1,4 glucan + ADP). Mutants isolated from maize endosperm as well as from Arabidopsis thaliana leaf that are deficient or lacking in starch strongly indicate that the ADPGlc pathway is the major if not the sole pathway in plants for synthesis of starch. Synthesis of the alpha-1,6 glucosidic branch points found in amylopectin are catalyzed by branching enzyme, EC2.4.1.18. Maize endosperm mutants, amylose extender, ae, lacking one of the branching enzyme isoforms contains a higher percentage of amylose and anomalous amylopectin than the normal maize endosperm thus indicating the physiological role of the branching enzyme. It has been shown that the plant ADPGlc synthetase activity, whether isolated from either leaf or non-photosynthetic reserve tissue, is activated by 3-phosphoglycerate (3PGA) and inhibited by orthophosphate (P(i)). The activator, 3PGA, at higher concentrations, can reverse or prevent inhibition by P(i). A number of studies have shown that the above biosynthetic enzymes are completely localized in the subcellular compartment where starch is formed; the chloroplasts in leaves and the amyloplasts in non-photosynthetic tissues. Recent data have also shown that the plant ADPGlc synthetase is composed of two different subunits, most probably, the products of two different genes. The subunit masses are 51 and 54 kDa for the spinach leaf and Arabidopsis enzymes, 60 and 55 kDa for the maize endosperm enzyme and 50 and 51 kDa for the potato tuber enzyme. The activator binding site of the smaller subunit of the spinach leaf enzyme has been elucidated with respect to the amino acid, (epsilon-amino group of lysyl residue) and the amino acid sequence surrounding it. It is localized near the carboxyl-terminal of the peptide. Similar sequences have been observed for subunits of the ADPGlc synthetases of at least 5 different plants, from photosynthetic or non-photosynthetic tissue as determined from amino acid sequences deduced from nucleotide sequences of cDNA clones. Using 8-azido ATP as a photoaffinity analogue, it has been shown that the larger subunit of the spinach leaf enzyme participates in the binding of the substrate. Other experiments are underway to determine the amino acid sequence of the substrate site. cDNA clones of the smaller subunit have been isolated from rice endosperm, spinach leaf and potato tuber and appear to be coding for the smaller subunit. At present a cDNA clone for the larger subunit has not been isolated put partial amino acid sequence analysis of the spinach leaf 54 kDa subunit indicates approximately a 30% identity with the 51 kDa subunit.