CHARACTERIZATION OF THE SUBSTRATE-SPECIFICITY OF SUCROSE-PHOSPHATE SYNTHASE PROTEIN-KINASE

Sucrose-phosphate synthase (SPS; EC 2.4.1.14) is regulated by reversib le protein phosphorylation. When the enzyme is phosphorylated it is in activated and can be reactivated by removal of phosphate. The major re gulatory phosphorylation site is known to be Ser(158) in the spinach-l eaf enzyme, and two protein kinase activities have been resolved chrom atographically which phosphorylate SPS at this site in vitro. In this report, we use a set of synthetic peptide substrate analogs based on t he phosphorylation site sequence, and a set of Escherichia coli-expres sed 26-kDa fragments of spinach SPS which contain the site, to identif y the recognition elements that target the two protein kinases to Ser( 158). The major recognition element consists of basic residues at P-3 and P-6 relative to the phosphorylated serine. Comparison of the spina ch enzyme amino-acid sequence with two other plant species show conser vation of these amino acids and implies that these signals are also co nserved. We also present evidence that glucose-6-phosphate is not only an allosteric activator of SPS but also an inhibitor of SPS-protein k inase per se, thereby allowing it to act at both levels of SPS regulat ion. (C) 1995 Academic Press, Inc.