Purification, oligomerization state and malate sensitivity of maize leaf phosphoenolpyruvate carboxylase

Abstract

A method was developed for the purification of phosphoenolpyruvate carboxylase from darkened maize leaves so that the enzyme retained its sensitivity to inhibition by malate. The procedure depended on the prevention of proteolysis by the inclusion of chymostatin in the buffers used during the purification. The purified enzyme was indistinguishable from that in crude extracts as judged by native polyacrylamide-gel electrophoresis. SDS/polyacrylamide-gel electrophoresis followed by immunoblotting, and Superose 6 gel filtration. Gel-filtration studies showed that the purified enzyme and the enzyme in extracts of darkened or illuminated leaves showed a concentration-dependent dissociation of tetrameric into dimeric forms. Purified phosphoenolpyruvate carboxylase and enzyme in crude extracts from darkened leaves were equally sensitive to inhibition by malate (Ki approx. 0.30 mM) under conditions where it existed in the tetrameric or dimeric forms, but the enzyme in crude extracts from illuminated leaves was less sensitive to malate inhibition (Ki approx. 0.95 mM) whether it was present as a tetramer or as a dimer. It is concluded that changes in the oligomerization state of phosphoenolpyruvate carboxylase are not directly involved in its regulation by light.

Evolution of the Enzymatic Characteristics of C4Phosphoenol Pyruvate Carboxylase

“…This discrepancy may be due to the lack of consensus in the isolation procedure of P-pyruvate carboxylases. It has be shown that P-pyruvate carboxylases are very susceptible to proteolysis of a small part of the N-terminus (McNaughton et al, 1989;Carter et al, 1991). This part of the enzyme harbours the serine residue shown to be the site of reversible phosphorylation.…”

Posttranslational Modification of Maize Chloroplast Pyruvate Orthophosphate Dikinase Reveals the Precise Regulatory Mechanism of Its Enzymatic Activity

“…This difference may reflect potential proteolysis at the N terminus during protein purification. Indeed, rapid proteolytic cleavage during the isolation of other key C 4 pathway enzymes, namely PEP carboxykinase and PEP carboxylase, provides a precedent for this possibility (McNaughton et al, 1989;Walker and Leegood, 1995;Walker et al, 1997). Using 2DGE and MS, we also identified the N termini of CyPPDKZm1 and CyPPDKZm2 in 25-d-old maize endosperm where the two proteins were highly expressed (Supplemental Fig.…”

Kinetic Analysis of the Non-Phosphorylated, in Vitro Phosphorylated, and Phosphorylation-Site-Mutant (Asp8) Forms of Intact Recombinant C4 Phosphoenolpyruvate Carboxylase from Sorghum

“…It has been reported that maize (C 4 [13]) or Mesembryunthemum crystallinum (inducible CAM [141) P-pyruvate carboxylase which had been purified in the absence of the proteinase inhibitor chymostatin had lost a peptide fragment from its N-or C-terminus, in addition to its malate sensitivity and ability to be phosphorylated in vitro. Therefore, it has been suggested that the time required for enzyme purification may profoundly influence the regulatory properties of plant P-pyruvate carboxylase [4].…”

“…Thus, limited proteolysis of this N-terminal regulatory domain during enzyme preparation may be responsible for much of the observed variation in malate sensitivity (cf. [4,13,14]). Indeed, although many detailed kinetic studies have been performed on C 4 and CAM Ppyruvate carboxylase (e.g.…”

“…Indeed, although many detailed kinetic studies have been performed on C 4 and CAM Ppyruvate carboxylase (e.g. [16][17][18][19][20][21]), the documented effect of proteolysis during enzyme preparation [11,[13][14][15] and the relatively low malate sensitivity of P-pyruvate carboxylase often observed in these previous studies [16][17][18][19] suggest that the enzyme used in these prior investigations was partially or completely truncated at its N-terminus.…”