SITE-DIRECTED MUTAGENESIS OF MAIZE RECOMBINANT C-4-PYRUVATE, ORTHOPHOSPHATE DIKINASE AT THE PHOSPHORYLATABLE TARGET THREONINE RESIDUE

A key regulatory enzyme of the C-4-photosynthetic pathway is stromal p yruvate,orthophosphate dikinase (PPDK, EC 2.7.9.1). As a pivotal enzym e in the C-4 pathway, it undergoes diurnal light-dark regulation of ac tivity which is mediated by a single bifunctional regulatory protein ( RP). RP specifically inactivates PPDK in the dark by an ADP-dependent phosphorylation of an active-site Thr residue (Thr-456 in maize). Conv ersely, RP activates inactive PPDK in the Light by phosphorolytic deph osphorylation of this target Thr-P residue. We have employed a His-tag ged maize recombinant C-4 PPDK for directed mutagenesis of this active -site regulatory Thr. Three such mutants (T456V, T456S, T456D) were an alyzed with respect to overall catalysis and regulation by exogenous m aize RP, Substitution with Val and Ser at this position does not affec t overall catalysis, whereas Asp abolishes enzyme activity. With respe ct to regulation by RP, it was found that Ser can effectively substitu te for the wild-type Thr residue in that mutant enzyme is phosphorylat ed and inactivated by RP. The T456V mutant, however, could not be phos phorylated and was, thus, resistant to ADP-dependent inactivation by R P. (C) 1997 Federation of European Biochemical Societies.