Journal
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Volume 375, Issue 1, Pages 165-170Publisher
ELSEVIER SCIENCE INC
DOI: 10.1006/abbi.1999.1651
Keywords
pyruvate; Pi dikinase (PPDK); site-directed mutagenesis; protein phosphorylation; regulatory protein; C-4 photosynthesis; maize (Zea mays)
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In C-4 plants such as maize, pyruvate,orthophosphate dikinase (PPDK) catalyzes the regeneration of the initial carboxylation substrate during C-4 photosynthesis, The primary catalytic residue, His-458 (maize C-4 PPDK), is involved in the ultimate transfer of the beta-phosphate from ATP to pyruvate, C-4 PPDK activity undergoes light-dark regulation in vivo by reversible phosphorylation of a nearby active-site residue (Thr-456) by a single bifunctional regulatory protein (RP). Using site-directed mutagenesis of maize recombinant C-4 dikinase, are made substitutions at the catalytic His residue (H458N) and at this regulatory target Thr (T456E, T456Y, T456F). Each of these affinity-purified mutant enzymes was assayed for changes in dikinase activity. As expected, substituting His-458 with Asn results in a catalytically incompetent enzyme, Substitutions of the Thr-456 residue with Tyr and Phe reduced activity by about 94 and 99%, respectively. Insertion of Glu at this position completely abolished activity, presumably by the introduction of negative charge proximal to the catalytic His. Furthermore, neither the T456Y nor inactive H458N mutant enzyme was phosphorylated in vitro by RP, The 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 work showed that RP effectively phosphorylated Ser inserted at position 456, The 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 intermediate 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.
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