Further analysis of maize C-4 pyruvate,orthophosphate dikinase phosphorylation by its bifunctional regulatory protein using selective substitutions of the regulatory Thr-456 and catalytic His-458 residues

In C-4 plants such as maize, pyruvate,orthophosphate dikinase (PPDK) cataly zes the regeneration of the initial carboxylation substrate during C-4 phot osynthesis, The primary catalytic residue, His-458 (maize C-4 PPDK), is inv olved in the ultimate transfer of the beta-phosphate from ATP to pyruvate, C-4 PPDK activity undergoes light-dark regulation in vivo by reversible pho sphorylation of a nearby active-site residue (Thr-456) by a single bifuncti onal regulatory protein (RP). Using site-directed mutagenesis of maize reco mbinant C-4 dikinase, are made substitutions at the catalytic His residue ( H458N) and at this regulatory target Thr (T456E, T456Y, T456F). Each of the se affinity-purified mutant enzymes was assayed for changes in dikinase act ivity. As expected, substituting His-458 with Asn results in a catalyticall y incompetent enzyme, Substitutions of the Thr-456 residue with Tyr and Phe reduced activity by about 94 and 99%, respectively. Insertion of Glu at th is position completely abolished activity, presumably by the introduction o f negative charge proximal to the catalytic His. Furthermore, neither the T 456Y nor inactive H458N mutant enzyme was phosphorylated in vitro by RP, Th e inability of the former to serve as a phosphorylation substrate indicates that RP is functionally a member of the Ser/Thr family of protein kinases rather than a "dual-specificity" Ser-Thr/Tyr kinase, since our previous wor k showed that RP effectively phosphorylated Ser inserted at position 456, T he inability of RP to phosphorylate its native target Thr residue when Asn is substituted for His-458 documents that RP requires the His-P catalytic i ntermediate form of PPDK as its protein substrate. For these latter studies , synthetic phosphopeptide-directed antibodies specific for the Thr(456)-P form of maize C-4 PPDK were developed and characterized. (C) 2000 Academic Press.