Site-directed mutagenesis of serine 158 demonstrates its role in spinach leaf sucrose-phosphate synthase modulation

Site-directed mutagenesis of spinach sucrose-phosphate synthase (SPS) was p erformed to investigate the role of Ser158 in the modulation of spinach lea f SPS. Tobacco plants expressing the spinach wild-type (WT), S158A, S158T a nd S157F/S158E SPS transgenes were produced. Expression of transgenes appea red not to reduce expression of the tobacco host SPS. SPS activity in the W T and the S158T SPS transgenics showed light/dark modulation, whereas the S 158A and S157F/S158E mutants were not similarly light/dark modulated: the S 158A mutant enzyme was not inactivated in the dark, and the S157F/S158E was not activated in the light. The inability to modulate the activity of the S158A mutant enzyme by protein phosphorylation was demonstrated in vitro. T he WT spinach enzyme immunopurified from dark transgenic tobacco leaves had a low initial activation state, and could be activated by PP2A and subsequ ently inactivated by SPS-kinase plus ATP. Rapid purification of the S158A m utant enzyme from dark leaves of transgenic plants using spinach-specific m onoclonal antibodies yielded enzyme that had a high initial activation stat e, and pre-incubation with leaf PP2A or ATP plus SPS-kinase (the PKIII enzy me) caused little modulation of activity. The results demonstrate the regul atory significance of Ser158 as the major site responsible for dark inactiv ation of spinach SPS in vivo, and indicate that the significance of phospho rylation is the introduction of a negative charge at the Ser158 position.