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Site‐directed mutagenesis of maize recombinant C 4 ‐pyruvate,orthophosphate dikinase at the phosphorylatable target threonine residue
Author(s) -
Chastain Chris J.,
Lee Mary E.,
Moorman Molly A.,
Shameekumar Patil,
Chollet Raymond
Publication year - 1997
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(97)00884-3
Subject(s) - biochemistry , dephosphorylation , site directed mutagenesis , mutant , phosphorylation , enzyme , chemistry , residue (chemistry) , threonine , saturated mutagenesis , active site , alanine , recombinant dna , mutagenesis , wild type , serine , phosphatase , amino acid , gene
A key regulatory enzyme of the C 4 ‐photosynthetic pathway is stromal pyruvate,orthophosphate dikinase (PPDK, EC 2.7.9.1). As a pivotal enzyme in the C 4 pathway, it undergoes diurnal light–dark regulation of activity 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). Conversely, RP activates inactive PPDK in the light by phosphorolytic dephosphorylation of this target Thr‐P residue. We have employed a His‐tagged maize recombinant C 4 PPDK for directed mutagenesis of this active‐site regulatory Thr. Three such mutants (T456V, T456S, T456D) were analyzed with respect to overall catalysis and regulation by exogenous maize RP. Substitution with Val and Ser at this position does not affect overall catalysis, whereas Asp abolishes enzyme activity. With respect to regulation by RP, it was found that Ser can effectively substitute for the wild‐type Thr residue in that mutant enzyme is phosphorylated and inactivated by RP. The T456V mutant, however, could not be phosphorylated and was, thus, resistant to ADP‐dependent inactivation by RP.